Targeted synthetic nanocarriers with ph sensitive release of immunostimulatory agents

ABSTRACT

This invention relates to compositions, and related methods, of synthetic nanocarriers that target sites of action in cells, such as antigen presenting cells (APCs), and comprise immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner. Also disclosed are compositions and methods relating to synthetic nanocarriers that encapsulate labile immunomodulatory agents that dissociate from the synthetic nanocarriers in a pH sensitive manner.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/788,260, filed May 26, 2010, now pending, which claims the benefitunder 35 U.S.C. §119 of U.S. Provisional Application Nos. 61/217,129,61/217,117, 61/217,124, and 61/217,116, each filed May 27, 2009, thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to compositions, and related methods, ofsynthetic nanocarriers that target sites of action in cells, such asantigen presenting cells (APCs), and comprise immunomodulatory agentsthat dissociate from the synthetic nanocarriers in a pH sensitivemanner. The invention additionally relates to protection of labileimmunomodulatory agents by means of their encapsulation in syntheticnanocarriers.

BACKGROUND

Immunomodulatory agents are used to produce immune responses insubjects. Stimulation of the immune system, which includes stimulationof either or both innate immunity and adaptive immunity, is a complexphenomenon that can result in either protective or adverse physiologicoutcomes for the host. In recent years there has been increased interestin the mechanisms underlying innate immunity, which is believed toinitiate and support adaptive immunity. This interest has been fueled inpart by the recent discovery of a family of highly conserved patternrecognition receptor proteins known as Toll-like receptors (TLRs)believed to be involved in innate immunity as receptors forpathogen-associated molecular patterns (PAMPs).

Compositions and methods useful for modulating innate immunity aretherefore of great interest, as they may affect therapeutic approachesto conditions involving inflammation, allergy, asthma, infection,cancer, and immunodeficiency, etc.

It is at times advantageous to couple such agents to delivery vehicles.However, information regarding how the release of such agents,especially labile immunomodulatory agents, from delivery vehicles can becontrolled and what kind of release provides for optimal in vivo effectsis lacking.

There is a need for new delivery vehicles for deliveringimmunomodulatory agents that allow for optimal release as well asrelated methods.

SUMMARY OF THE INVENTION

Aspects of the invention relate to compositions comprising syntheticnanocarriers that comprise an immunomodulatory agent coupled to thesynthetic nanocarrier, wherein the immunomodulatory agent dissociatesfrom the synthetic nanocarrier according to the following relationship:IArel(4.5)₂₄%/IArel(7.4)₂₄%≧1.2, wherein IArel(4.5)₂₄% is defined as aweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hoursdivided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=4.5 for 24 hours plus a weight of immunomodulatory agentretained in the synthetic nanocarrier upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours,expressed as weight percent, and taken as an average across a sample ofthe synthetic nanocarriers, and wherein IArel(7.4)₂₄% is defined as aweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hoursdivided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=7.4 for 24 hours plus a weight of immunomodulatory agentretained in the synthetic nanocarrier upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours,expressed as weight percent, and taken as an average across a sample ofthe synthetic nanocarriers.

In some embodiments, the immunomodulatory agent is coupled to thesynthetic nanocarrier via an immunomodulatory agent coupling moiety. Incertain embodiments, the immunomodulatory agent is encapsulated withinthe synthetic nanocarrier. In some embodiments, the immunomodulatoryagent comprises a labile immunomodulatory agent such as animidazoquinoline, an adenine derivative, or an oligonucleotide thatcomprises 5′-CG-3′, wherein C is unmethylated and wherein theoligonucleotide comprises a backbone comprising one or more unstabilizedinternucleotide linkages. In certain embodiments, the imidazoquinolinecomprises an imidazoquinoline amine, an imidazopyridine amine, a6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine,imiquimod, or resiquimod.

In some embodiments, the oligonucleotide's backbone comprises nostabilizing chemical modifications that function to stabilize thebackbone under physiological conditions. In some embodiments, theoligonucleotide's backbone comprises a backbone that is not modified toincorporate phosphorothioate stabilizing chemical modifications. In someembodiments, the immunomodulatory agent is an adjuvant. In certainembodiments, the adjuvant comprises a Toll-like receptor (TLR) agonistsuch as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8 agonist,or a TLR 9 agonist.

In some embodiments, the TLR agonist is an immunostimulatory nucleicacid such as an immunostimulatory DNA or immunostimulatory RNA. Incertain embodiments, the immunostimulatory nucleic acid is aCpG-containing immunostimulatory nucleic acid that comprises one or morestabilizing chemical modifications that function to stabilize thebackbone under physiological conditions. In some embodiments, theadjuvant comprises a universal T-cell antigen.

In some embodiments, the synthetic nanocarriers further comprise a Bcell antigen and/or a T cell antigen. In certain embodiments, thesynthetic nanocarriers further comprise an antigen presenting cell (APC)targeting feature. In some embodiments, the synthetic nanocarrierscomprise one or more biodegradable polymers. In some embodiments, theimmunomodulatory agent is coupled to the one or more biodegradablepolymers via the immunomodulatory agent coupling moiety. In certainembodiments, the biodegradable polymer comprises poly(lactide),poly(glycolide), or poly(lactide-co-glycolide).

In some embodiments, the biodegradable polymers have a weight averagemolecular weight ranging from 800 Daltons to 10,000 Daltons, asdetermined using gel permeation chromatography. In certain embodiments,the immunomodulatory agent coupling moiety comprises an amide bond. Insome embodiments, the immunomodulatory agent coupling moiety comprisesan ester bond.

In some embodiments, the synthetic nanocarriers comprise lipid-basednanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles, peptide or protein-based particles, nanoparticlesthat comprise a combination of nanomaterials, spheroidal nanoparticles,cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles,cylindrical nanoparticles, or toroidal nanoparticles.

Aspects of the invention relate to compositions comprising syntheticnanocarriers that comprise an immunomodulatory agent coupled to thesynthetic nanocarrier, wherein the immunomodulatory agent dissociatesfrom the synthetic nanocarrier according to the following relationship:IA(4.5)₂₄/IA(4.5)₆≧1.2, wherein IA(4.5)₂₄ is defined as a weight ofimmunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hourstaken as an average across a sample of the synthetic nanocarriers, andwherein IA(4.5)₆ is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for 6 hours taken as an average across asample of the synthetic nanocarriers.

In some embodiments, the immunomodulatory agent comprises a labileimmunomodulatory agent encapsulated within the synthetic nanocarrier. Incertain embodiments, the labile immunomodulatory agent comprises animidazoquinoline, an adenine derivative, or an oligonucleotide thatcomprises 5′-CG-3′, wherein C is unmethylated and wherein theoligonucleotide comprises a backbone comprising one or more unstabilizedinternucleotide linkages. In certain embodiments, the imidazoquinolinecomprises an imidazoquinoline amine, an imidazopyridine amine, a6,7-fused cycloalkylimidazopyridine amine, an imidazoquinoline amine,imiquimod, or resiquimod. In some embodiments, the oligonucleotide'sbackbone comprises no stabilizing chemical modifications that functionto stabilize the backbone under physiological conditions. In someembodiments, the oligonucleotide's backbone comprises a backbone that isnot modified to incorporate phosphorothioate stabilizing chemicalmodifications.

Further aspects of the invention relate to compositions comprisingsynthetic nanocarriers that comprise an immunomodulatory agent coupledto the synthetic nanocarrier, wherein the immunomodulatory agentdissociates from the synthetic nanocarrier according to the followingrelationship: 6≦IA(4.5)₂₄/IA(4.5)₆≧1.2, wherein IA(4.5)₂₄ is defined asa weight of immunomodulatory agent released upon exposure of thesynthetic nanocarrier to an in vitro aqueous environment at a pH=4.5 for24 hours taken as an average across a sample of the syntheticnanocarriers, and wherein IA(4.5)₆ is defined as a weight ofimmunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 6 hourstaken as an average across a sample of the synthetic nanocarriers.

In some embodiments, the immunomodulatory agent comprises a labileimmunomodulatory agent encapsulated within the synthetic nanocarrier. Insome embodiments, the labile immunomodulatory agent comprises animidazoquinoline, an adenine derivative, or an oligonucleotide thatcomprises 5′-CG-3′, wherein C is unmethylated and wherein theoligonucleotide comprises a backbone comprising one or more unstabilizedinternucleotide linkages. In certain embodiments, the imidazoquinolinecomprises an imidazoquinoline amine, an imidazopyridine amine, a6,7-fused cycloalkylimidazopyridine amine, a imidazoquinoline amine,imiquimod, or resiquimod.

In some embodiments, the oligonucleotide's backbone comprises nostabilizing chemical modifications that function to stabilize thebackbone under physiological conditions. In some embodiments, theoligonucleotide's backbone comprises a backbone that is not modified toincorporate phosphorothioate stabilizing chemical modifications. Incertain embodiments, the immunomodulatory agent is coupled to thesynthetic nanocarrier via an immunomodulatory agent coupling moiety. Insome embodiments, the immunomodulatory agent is encapsulated within thesynthetic nanocarrier.

In some embodiments, the immunomodulatory agent is an adjuvant. Incertain embodiments, the adjuvant comprises a Toll-like receptor (TLR)agonist such as a TLR 3 agonist, TLR 7 agonist, TLR 8 agonist, TLR 7/8agonist, or a TLR 9 agonist. In certain embodiments, the TLR agonist isan immunostimulatory nucleic acid such as an immunostimulatory DNA orimmunostimulatory RNA.

In some embodiments, the immunostimulatory nucleic acid is aCpG-containing immunostimulatory nucleic acid that comprises one or morestabilizing chemical modifications that function to stabilize thebackbone under physiological conditions. In certain embodiments, theadjuvant comprises a universal T-cell antigen. In some embodiments, thesynthetic nanocarriers further comprise a B cell antigen and/or a T cellantigen.

In some embodiments, the synthetic nanocarriers further comprise anantigen presenting cell (APC) targeting feature. In certain embodiments,the synthetic nanocarriers comprise one or more biodegradable polymers.In some embodiments, the immunomodulatory agent is coupled to the one ormore biodegradable polymers via the immunomodulatory agent couplingmoiety. In certain embodiments, the biodegradable polymer comprisespoly(lactide), poly(glycolide), or poly(lactide-co-glycolide).

In some embodiments, the biodegradable polymers have a weight averagemolecular weight ranging from 800 Daltons to 10,000 Daltons, asdetermined using gel permeation chromatography. In certain embodiments,the immunomodulatory agent coupling moiety comprises an amide bond. Insome embodiments, the immunomodulatory agent coupling moiety comprisesan ester bond.

In some embodiments, the synthetic nanocarriers comprise lipid-basednanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles, peptide or protein-based particles, nanoparticlesthat comprise a combination of nanomaterials, spheroidal nanoparticles,cubic nanoparticles, pyramidal nanoparticles, oblong nanoparticles,cylindrical nanoparticles, or toroidal nanoparticles. In certainembodiments, compositions associated with the invention further comprisea pharmaceutically acceptable excipient.

Further aspects of the invention relate to compositions comprising avaccine comprising any of the compositions associated with theinvention.

Further aspects of the invention involve methods comprisingadministering any of the compositions associated with the invention to asubject. In some embodiments, the composition is in an amount effectiveto induce or enhance an immune response. In some embodiments, thesubject has cancer, an infectious disease, a non-autoimmune metabolicdisease, a degenerative disease, or an addiction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the release of resiquimod (R848) from syntheticnanocarrier formulations at pH 7.4, 37° C.

FIG. 2 demonstrates the release of R848 from synthetic nanocarrierformulations at pH 4.5, 37° C.

FIG. 3 demonstrates the release of R848 from synthetic nanocarrierformulations at pH 7.4 and pH 4.5 at 24 hours.

FIG. 4 shows the level of antibody induction by synthetic nanocarrierswith a CpG-containing immunostimulatory nucleic acid (Groups 2 and 3) ascompared to the level of antibody induction by synthetic nanocarrierswithout the CpG-containing immunostimulatory nucleic acid (Group 1).

FIG. 5 shows the level of antibody induction by synthetic nanocarriersthat release a phosphodiester, non-thioated CpG-containingimmunostimulatory nucleic acid or a thioated CpG-containingimmunostimulatory nucleic acid.

FIG. 6 shows the level of antibody induction by synthetic nanocarriersthat release R848 at different rates.

FIG. 7 shows the level of antibody induction by synthetic nanocarrierscarrying entrapped phosphodiester (PO) CpG, designated as NC-Nic/PO-CpG.

FIG. 8 shows the release of entrapped PO-CpG from nanocarriers at a pHof 4.5 versus pH 7.5. The data demonstrates that a labileimmunomodulatory agent, such as PO-CpG, is protected by encapsulationwithin a synthetic nanocarrier. Such a labile agent can be released at adesired site of action with a pH of 4.5 (e.g., in the endosome/lysosome)with low levels of release occurring at a pH of 7.4 (e.g., generally thepH outside of the endosome/lysosome).

DETAILED DESCRIPTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules, reference to “asolvent” includes a mixture of two or more such solvents, reference to“an adhesive” includes mixtures of two or more such materials, and thelike.

Introduction

This invention is useful in that it provides a way to releaseimmunomodulatory agents more directly at the sites of action in cells ofinterest, in particular antigen presenting cells, which would result inbeneficial immune response and/or reduce off-target effects andtoxicity, as the majority of the release of the immunomodulatory agentswould be at a site of action in the cells of interest. This is ofparticular interest for the delivery of adjuvants. The controlledrelease properties offer for the first time a controlled way ofdelivering immunomodulatory agents to the immune cells of interest andallow for a more precise intervention on the immune system, includingthe ability to release immunomodulatory agents over an extended period.All of this leads to a very tunable system to get the optimum release ofimmunomodulatory agent such that it will release primarily at a site ofaction in the desired cells.

The inventors have further recognized that coupling labileimmunomodulatory agents within the inventive synthetic nanocarriersthrough encapsulating the labile immunomodulatory agents within theinventive synthetic nanocarriers, and providing a controlled way ofdelivering labile immunomodulatory agents to immune cells of interest,preferably over an extended period, results in targeted delivery of thelabile immunomodulatory agents while minimizing off-target effects ofthe immunomodulatory agents, especially off-target effects associatedwith systemic administration of the immunomodulatory agents.Additionally, this approach can enhance the performance of labileimmunomodulatory agents having a short half-life of elimination thatotherwise might not have a desirable level of pharmacological activity.

In one embodiment, the invention relates to certain oligonucleotides.Recently, there have been a number of reports describing theimmunostimulatory effect of certain types of nucleic acid molecules,including CpG nucleic acids, GU rich ssRNA and double-stranded RNA. Ofnote, it was recently reported that Toll-like receptor 9 (TLR9)recognizes bacterial DNA and oligonucleotides containing a CpG motifwherein the cytosine is unmethylated. Hemmi H et al. (2000) Nature408:740-5; Bauer S. et al. (2001) Proc Natl Acad Sci USA 98:9237-42. Theeffects of CpG containing oligonucleotides on immune modulation havebeen described extensively in U.S. patents such as U.S. Pat. Nos.6,194,388; 6,207,646; 6,239,116; and 6,218,371, and publishedinternational patent applications, such as W098/37919, W098/40100,W098/52581, and W099/56755. The entire immunostimulatory nucleic acidcan be unmethylated or portions may be unmethylated but at least the Cof the 5′-CG-3′ must be unmethylated.

Natural DNA oligonucleotides contain phosphodiester linkages that arerapidly cleaved by nucleases found in the extracellular environment. Yu,D., et al., Potent CpG oligonucleotides containing phosphodiesterlinkages: in vitro and in vivo immunostimulatory properties. BiochemBiophys Res Commun, 2002. 297(1): p. 83-90 (“Yu et al.”); Heeg, K., etal., Structural requirements for uptake and recognition of CpGoligonucleotides. Int J Med Microbiol, 2008. 298(1-2): p. 33-8 (“Heeg etal.”). Such natural oligonucleotides may be considered labileimmunomodulatory agents. Accordingly, methods of chemically stabilizingthe linkages by replacing the phosphodiester linking group with aphosphorothioate group have been extensively reported in the literature.See U.S. Pat. No. 6,811,975—Phosphorothioate Oligonucleotides HavingModified Internucleoside Linkages.

Phosphorothioate CpG containing oligonucleotides have been administeredsystemically as vaccine adjuvants. Yu et al. However, systemicadministration of stabilized CpG oligonucleotides can result inoff-target immunostimulatory effects, such as general inflammation,non-specific activation of lymphocytes, and flu-like symptoms. Haas, T.,et al., Sequence independent interferon-alpha induction by multimerizedphosphodiester DNA depends on spatial regulation of Toll-like receptor-9activation in plasmacytoid dendritic cells. Immunology, 2009. 126(2): p.290-8 (“Haas et al.”). Accordingly, such oligonucleotides may beusefully incorporated in the practice of the present invention, as isdescribed in more detail below.

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide, together withrelated methods, a composition comprising: synthetic nanocarriers thatcomprise an immunomodulatory agent coupled to the synthetic nanocarrier;wherein the immunomodulatory agent, preferably a labile immunomodulatoryagent, dissociates from the synthetic nanocarrier according to thefollowing relationship:

IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2;

wherein IArel(4.5)_(t)% is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for t hours divided by the sum of theweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for t hoursplus a weight of immunomodulatory agent retained in the syntheticnanocarrier upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for t hours, expressed as weightpercent, and taken as an average across a sample of the syntheticnanocarriers; and wherein IArel(7.4)_(t)% is defined as a weight ofimmunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for t hoursdivided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=7.4 for t hours plus a weight of immunomodulatory agent retainedin the synthetic nanocarrier upon exposure of the synthetic nanocarrierto an in vitro aqueous environment at a pH=7.4 for t hours, expressed asweight percent, and taken as an average across a sample of the syntheticnanocarriers; and wherein t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or 30 hours.

In some embodiments, the immunomodulatory agent, preferably a labileimmunomodulatory agent, dissociates from the synthetic nanocarrieraccording to the following relationship:IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.3,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.4,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.5,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.6,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.7,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.8,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.9, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2.2,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2.5,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2.7, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧3,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧3.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧4,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧4.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧5,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧5.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧6,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧6.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧7,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧7.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧8,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧8.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧9,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧9.5, IArel(4.5)_(t)%/IArel(7.4)_(t)%≧10,IArel(4.5)_(t)%/IArel(7.4)_(t)%≧10.5, orIArel(4.5)_(t)%/IArel(7.4)_(t)%≧11, wherein IArel(4.5)_(t)%,IArel(7.4)_(t)%, and t are as defined above.

In other embodiments, the immunomodulatory agent, preferably a labileimmunomodulatory agent, dissociates from the synthetic nanocarrieraccording to the following relationship:2≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,2.5≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,3≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,3.5≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,4≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,4.5≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,5≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,6≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,7≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,8≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,9≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧1.2,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧2.5,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧3,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧3.5,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧4,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧4.5,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧5,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧6,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧7,10≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧8, 10≦IArel(4.5)_(t)%/IArel(7.4) %≧9,3≦IArel(4.5)_(t)%/IArel(7.4)_(t)% 2,4≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧3,5≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧4,6≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧5,7≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧6,8≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧7, or9≦IArel(4.5)_(t)%/IArel(7.4)_(t)%≧8, wherein IArel(4.5)_(t)%,IArel(7.4)_(t)%, and t are as defined above. In some embodiments, t is24 hours.

Accordingly, this invention relates to compositions and methodscomprising synthetic nanocarriers that release immunomodulatory agentsat significantly different rates at neutral and acidic pH. In deliveringimmunomodulatory agents, to have the most potent effect it is desirableto have the majority of the immunomodulatory agent released inside APCswhere they can have a desired effect. When immunomodulatory agents areinjected in free form, or when they are released from a syntheticnanoparticle outside the APCs, only a small portion of thatimmunomodulatory agent finds its way to the APCs, while the restdiffuses through the body, where the immune stimulation would be lessand may result in deleterious effects. The inventive syntheticnanocarriers provided herein are preferentially taken up by APCs. Uponbeing taken up by the APC, the synthetic nanocarriers are presumed to beendocytosed into an endosomal/lysosomal compartment where the pH becomesmore acidic, as opposed to the neutral pH outside the cells. Under theseconditions, the immunomodulatory agent exhibits a pH sensitivedissociation from the synthetic nanocarrier (e.g., from animmunomodulatory agent coupling moiety) and is released from thesynthetic nanocarrier. The immunomodulatory agent is then free tointeract with receptors associated with the endosome/lysosome andstimulate a desired immune response. The property of the inventivesynthetic nanocarriers of having lower release of immunomodulatoryagents at or about neutral pH, or in embodiments at or aboutphysiological pH (i.e., pH=7.4), but increased release at or about a pHof 4.5 is desirable for it targets the immunomodulatory agents to theendosomal/lysosomal compartment of APCs to which the syntheticnanocarriers target.

The immunomodulatory agents can be coupled to the synthetic nanocarriersby any of a number of methods. Generally, the coupling can be a resultof bonding between the immunomodulatory agent and the syntheticnanocarrier. This bonding can result in the immunomodulatory agent beingattached to the surface of the synthetic nanocarrier and/or containedwithin (encapsulated) the synthetic nanocarrier. In some embodiments,however, the immunomodulatory agent is encapsulated by the syntheticnanocarrier as a result of the structure of the synthetic nanocarrierrather than bonding to the synthetic nanocarrier.

When coupling occurs as a result of bonding between the immunomodulatoryagent and synthetic nanocarrier, the coupling occurs via animmunomodulatory agent coupling moiety. An immunomodulatory agentcoupling moiety can be any moiety through which an immunomodulatoryagent is bonded to a synthetic nanocarrier. Such moieties includecovalent bonds, such as an amide bond or ester bond, as well as separatemolecules that bond (covalently or non-covalently) the immunomodulatoryagent to the synthetic nanocarrier. Such molecules include linkers orpolymers or a unit thereof. For example, the immunomodulatory agentcoupling moiety can comprise a charged polymer to which animmunomodulatory agent (e.g., an immunostimulatory nucleic acid)electrostatically binds. As another example, the immunomodulatory agentcoupling moiety can comprise a polymer or unit thereof to which theimmunomodulatory agent is covalently bonded.

In some embodiments, the polymer or unit thereof comprises a polyester,polycarbonate, polyamide, or polyether, or unit thereof. In otherembodiments, the polymer or unit thereof comprises poly(ethylene glycol)(PEG), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), or a polycaprolactone, or unit thereof. In some embodiments, itis preferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or unit thereof, the polymer comprises ablock-co-polymer of a polyether and a biodegradable polymer such thatthe polymer is biodegradable. In other embodiments, the polymer does notsolely comprise a polyether or unit thereof, such as poly(ethyleneglycol) or unit thereof. The immunomodulatory agent coupling moiety asprovided herein, therefore, can comprise one of the aforementionedpolymers or a unit thereof (e.g., a lactide or glycolide).

In some embodiments, for use as part of a synthetic nanocarrier, thepolymer of the compounds or conjugates provided herein is insoluble inwater at pH=7.4 and at 25° C., is biodegradable, or both. In otherembodiments, the polymer is insoluble in water at pH=7.4 and at 25° C.but soluble at pH=4.5 and at 25° C. In still other embodiments, thepolymer is insoluble in water at pH=7.4 and at 25° C. but soluble atpH=4.5 and at 25° C. and biodegradable. In other embodiments, any of thepolymers provided herein can have a weight average molecular weight, asdetermined by gel permeation chromatography, of about 800 Da to 10,000Da (e.g., 2,000 Da).

In one embodiment, the immunomodulatory agent is an adjuvant, such as animidazoquinoline. Imidazoquinolines include compounds, such as imiquimodand resiquimod (also known as R848). Such adjuvants can be coupled to apolymer as provided above. As an example, resiquimod was conjugated topoly-lactic acid (PLA) polymer of −2000 Da. In in vitro release studies,such an embodiment demonstrated an increase in R848 release of 3- to6-fold when the pH was dropped from 7.4 to 4.5. Table 1 lists thecompositions of the particles tested. These included two formulationsthat encapsulated R848, 2 formulations with the PLA coupled covalentlyto R848 through the R848 amine, and four formulations with PLA coupledcovalently to R848 (via a ring opening method). In all formulations, therelease of R848 was significantly increased at the lower pH. Theencapsulated release rate is much faster than the conjugated releaserates, and there are also differences in release rates between theconjugation methods.

TABLE 1 Formulation Targets With A Covalent R848 Ova PLA- PLA-R848 R848peptide PEG- conjugate PLA (15-20K, Formulation load* load NIC type** BIR202H) Chemistry 1 E1.5% 1.1-2.2% 25% 75% 2 E1.5%++ 1.1-2.2% 25% 75% 3C75% 0.15-0.31% 25% Method 1 Amine 4 C75% 0.15-0.31% 25% Method 1 Amine5 C75% 0.15-0.31% 25% Method 5 ROP-hi MW 6 C75% 0.15-0.31% 25% Method 5ROP-lo MW 7 C50% 0.15-0.31% 25% Method 5 25% ROP-lo MW 8 C25% 0.15-0.31%25% Method 5 50% ROP-lo MW *C = covalent R848; E = encapsulation of R848

Although the above example was with PLA, immunomodulatory agents, suchas R848, can be coupled to other polymers or units thereof, such asthose provided above and elsewhere herein includingpolylactide-co-glycolide (PLGA) block co-polymer or unit thereof.Immunomodulatory agents, such as R848, can be coupled to such polymersor units thereof by an amide or ester bond. Examples of methods foreffecting such coupling are provided elsewhere herein and in theEXAMPLES.

The inventors have also unexpectedly discovered that it is possible toprovide, together with related methods, a composition comprising:

synthetic nanocarriers that comprise an immunomodulatory agent coupledto the synthetic nanocarrier; wherein the immunomodulatory agent,preferably a labile immunomodulatory agent, dissociates from thesynthetic nanocarrier according to the following relationship:

IA(4.5)_(t1) /IA(4.5)_(t2)≧1.2;

wherein IA(4.5)_(t1) is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for t1 hours taken as an average acrossa sample of the synthetic nanocarriers; and wherein IA(4.5)_(t2) isdefined as a weight of immunomodulatory agent released upon exposure ofthe synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5for t2 hours taken as an average across a sample of the syntheticnanocarriers; and wherein t1 is 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28 or 30 hours; t2 is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, or 28 hours; and t1>t2. In some embodiments, t1 is 24 hours, and t2is 6 hours.

In some embodiments, the immunomodulatory agent, preferably a labileimmunomodulatory agent, dissociates from the synthetic nanocarrieraccording to the following relationship: IA(4.5)_(t1)/IA(4.5)_(t2)≧1.5,IA(4.5)_(t1)/IA(4.5)_(t2)≧2, IA(4.5)_(t1)/IA(4.5)_(t2)≧2.5,IA(4.5)_(t1)/IA(4.5)_(t2)≧3, IA(4.5)_(t1)/IA(4.5)_(t2)≧3.5,IA(4.5)_(t1)/IA(4.5)_(t2)≧4, IA(4.5)_(t1)/IA(4.5)_(t2)≧4.5,IA(4.5)_(t1)/IA(4.5)_(t2)≧5, IA(4.5)_(t1)/IA(4.5)_(t2)≧6,IA(4.5)_(t1)/IA(4.5)_(t2)≧7, IA(4.5)_(t1)/IA(4.5)_(t2)≧8,IA(4.5)_(t1)/IA(4.5)_(t2)≧9, or IA(4.5)_(t1)/IA(4.5)_(t2)≧10; whereinIA(4.5)_(t1), IA(4.5)_(t2), t1, and t2 are as defined above. In someembodiments, t1 is 24 hours, and t2 is 6 hours.

In other embodiments, the immunomodulatory agent, preferably a labileimmunomodulatory agent, dissociates from the synthetic nanocarrieraccording to the following relationship:10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2,10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2.5, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧3,10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧3.5, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧4,10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧4.5, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧5,10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧6, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧7,10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧8, 10≦IA(4.5)_(t1)/IA(4.5)_(t2)≧9,9≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 8≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,7≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 4.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,4≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 3.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,3≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 2.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,2≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 1.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,3≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2, 4≦IA(4.5)_(t1)/IA(4.5)_(t2)≧3,5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧4, 6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧5,7≦IA(4.5)_(t1)/IA(4.5)_(t2)≧6, 8≦IA(4.5)_(t1)/IA(4.5)_(t2)≧7, or9≦IA(4.5)_(t1)/IA(4.5)_(t2)≧8; wherein IA(4.5)_(t1), IA(4.5)_(t2), t1,and t2 are as defined above. In some embodiments, t1 is 24 hours, and t2is 6 hours.

Inventive synthetic nanocarriers have also been shown to exhibit theproperty of augmenting a humoral immune response to a specific antigen.Such augmented humoral immune response has been found to be elevated, insome embodiments, with faster release of immunomodulatory agent. In oneembodiment, the immunomodulatory agent is a CpG-containingimmunostimulatory nucleic acid, and the CpG-containing immunostimulatorynucleic acid is encapsulated within a synthetic nanocarrier. In in vitrostudies, described further below in the EXAMPLES, it was found thatoptimal release of the CpG-containing immunostimulatory nucleic acidsfrom synthetic nanocarriers produced an elevated humoral immune responseto nicotine, which was also coupled to the synthetic nanocarriers. Insome embodiments, such optimal release was found to better augment anantibody response to an antigen.

Optimal release is the dissociation of the immunomodulatory agent fromthe synthetic nanocarrier that produces the best levels of desiredeffect(s). In some embodiments, the desired effect is an immediateimmune response of a desired level (i.e., one that occurs soon after theadministration of the synthetic nanocarrier). Generally, an immediateimmune response is one measured on the order of seconds, minutes, or afew hours. In other embodiments, the desired effect is an immuneresponse of a desired level that occurs after a few hours. In stillother embodiments, the desired effect is an immune response of a desiredlevel that is sustained for an extended period of time, such as for 1,2, 5, 10, 15 or more hours. In other embodiments, the extended period oftime is for 1, 2, 5, 10, 15, 20, 25, 30 or more days. In furtherembodiments, the extended period of time is for 1, 2, 5, 10 or moremonths. In further embodiments, the extended period of time is for 1, 2,5, 10 or more years. In some embodiments, a composition of syntheticnanocarriers that provides optimal release is one wherein theimmunomodulatory agent dissociates from the synthetic nanocarrieraccording to one of the above relationships.

In embodiments, an immunomodulatory agent, preferably a labileimmunomodulatory agent, that dissociates from the synthetic nanocarrierat an intermediate rate satisfies the following relationship:6≦IA(4.5)_(t1)/IA(4.5)_(t2)≦1.2, 5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,4≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 3≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2,2≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.2, 6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2,6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2.5, 6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧3,6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧3.5, 6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧4,6≦IA(4.5)_(t1)/IA(4.5)_(t2)≧5, 4≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.5,3.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.5, 3≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.5,2.5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧1.5, 5≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2,4≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2, or 3≦IA(4.5)_(t1)/IA(4.5)_(t2)≧2; whereinIA(4.5)_(t1), IA(4.5)_(t2), t1, and t2 are as defined above. In someembodiments, t1 is 24 hours, and t2 is 6 hours.

As another example, resiquimod was encapsulated within a syntheticnanocarrier. In in vitro studies, described further below in theEXAMPLES, it was found that resiquimod contained in the syntheticnanocarriers augmented humoral immune response against nicotine alsocoupled to the synthetic nanocarriers. It was also found that anintermediate release of the resiquimod from the synthetic nanocarrierswas optimal, as it resulted in a higher level of antibody induction thanfast or slow release of the resiquimod.

Accordingly, the synthetic nanocarriers provided herein can alsocomprise one or more antigens. The antigens can be B cell antigens or Tcell antigens or a combination of both. Such antigens can be coupled tothe synthetic nanocarriers such that they are present on the surface ofthe synthetic nanocarriers, encapsulated within the nanocarriers orboth, in some embodiments.

In embodiments, the immunomodulatory agent augments an immune responseto such an antigen. As mentioned above, the antigen can also be coupledto the synthetic nanocarriers. In other embodiments, however such asantigen is not coupled to the synthetic nanocarriers. In some of theseembodiments, such an antigen can be coadministered to a subject. Instill other of these embodiments, such an antigen is not coadministeredto the subject.

DEFINITIONS

“Adjuvant” means an agent that does not constitute a specific antigen,but boosts the strength and longevity of immune response to an antigen.Such adjuvants may include, but are not limited to stimulators ofpattern recognition receptors, such as Toll-like receptors, RIG-1 andNOD-like receptors (NLR), mineral salts, such as alum, alum combinedwith monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihiacoli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexnerior specifically with MPL® (AS04), MPL A of above-mentioned bacteriaseparately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX™,emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02(QS21+squalene+MPL®) liposomes and liposomal formulations such as AS01,synthesized or specifically prepared microparticles and microcarrierssuch as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae,Chlamydia trachomatis and others, or chitosan particles, depot-formingagents, such as Pluronic® block co-polymers, specifically modified orprepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide4-phosphates, such as RC529, or proteins, such as bacterial toxoids ortoxin fragments. In embodiments, adjuvants comprise agonists for patternrecognition receptors (PRR), including, but not limited to Toll-LikeReceptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/orcombinations thereof. In other embodiments, adjuvants comprise agonistsfor Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, oragonists for Toll-Like Receptor 9; preferably the recited adjuvantscomprise imidazoquinolines; such as resiquimod (also known as R848);adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381(Sumitomo Pharmaceutical Company); immunostimulatory DNA; orimmunostimulatory RNA. In specific embodiments, synthetic nanocarriersincorporate as adjuvants compounds that are agonists for toll-likereceptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al.,including but not limited to imidazoquinoline amines, imidazopyridineamines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridgedimidazoquinoline amines. Preferred adjuvants comprise imiquimod andresiquimod. In specific embodiments, an adjuvant may be an agonist forthe DC surface molecule CD40. In certain embodiments, a syntheticnanocarrier incorporates an adjuvant that promotes DC maturation (neededfor effective priming of naive T cells) and the production of cytokines,such as type I interferons, which in turn stimulate antibody andcytotoxic immune responses against desired antigen. In embodiments,adjuvants also may comprise immunostimulatory RNA molecules, such as butnot limited to dsRNA or poly I:C (a TLR3 stimulant), and/or thosedisclosed in F. Heil et al., “Species-Specific Recognition ofSingle-Stranded RNA via Toll-like Receptor 7 and 8” Science 303(5663),1526-1529 (2004); J. Vollmer et al., “Immune modulation by chemicallymodified ribonucleosides and oligoribonucleotides” WO 2008033432 A2; A.Forsbach et al., “Immunostimulatory oligoribonucleotides containingspecific sequence motif(s) and targeting the Toll-like receptor 8pathway” WO 2007062107 A2; E. Uhlmann et al., “Modifiedoligoribonucleotide analogs with enhanced immunostimulatory activity”U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al.,“Immunostimulatory viral RNA oligonucleotides and use for treatingcancer and infections” WO 2005097993 A2; G. Lipford et al.,“Immunostimulatory G,U-containing oligoribonucleotides, compositions,and screening methods” WO 2003086280 A2. In some embodiments, anadjuvant may be a TLR-4 agonist, such as bacterial lipopolysacccharide(LPS), VSV-G, and/or HMGB-1. In some embodiments, adjuvants may compriseTLR-5 agonists, such as flagellin, or portions or derivatives thereof,including but not limited to those disclosed in U.S. Pat. Nos.6,130,082, 6,585,980, and 7,192,725. In specific embodiments, syntheticnanocarriers incorporate a ligand for Toll-like receptor (TLR)-9, suchas immunostimulatory oligonucleotide molecules comprising 5′-CG-3′motifs, wherein the C is unmethylated, which induce type I interferonsecretion, and stimulate T and B cell activation leading to increasedantibody production and cytotoxic T cell responses (Krieg et al., CpGmotifs in bacterial DNA trigger direct B cell activation. Nature. 1995.374:546-549; Chu et al. CpG oligodeoxynucleotides act as adjuvants thatswitch on T helper 1 (Th1) immunity. J. Exp. Med. 1997. 186:1623-1631;Lipford et al. CpG-containing synthetic oligonucleotides promote B andcytotoxic T cell responses to protein antigen: a new class of vaccineadjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al.Immunostimulatory DNA sequences function as T helper-1-promotingadjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a potentenhancer of specific immunity in mice immunized with recombinanthepatitis B surface antigen. J. Immunol. 1998. 160:870-876; Lipford etal., Bacterial DNA as immune cell activator. Trends Microbiol. 1998.6:496-500. In some embodiments, adjuvants may be proinflammatory stimulireleased from necrotic cells (e.g., urate crystals). In someembodiments, adjuvants may be activated components of the complementcascade (e.g., CD21, CD35, etc.). In some embodiments, adjuvants may beactivated components of immune complexes. The adjuvants also includecomplement receptor agonists, such as a molecule that binds to CD21 orCD35. In some embodiments, the complement receptor agonist inducesendogenous complement opsonization of the synthetic nanocarrier. In someembodiments, adjuvants are cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, the cytokine receptoragonist is a small molecule, antibody, fusion protein, or aptamer.

“Administering” or “administration” means providing a drug to a patientin a manner that is pharmacologically useful.

“APC targeting feature” means one or more portions of which theinventive synthetic nanocarriers are comprised that target the syntheticnanocarriers to professional antigen presenting cells (“APCs”), such asbut not limited to dendritic cells, SCS macrophages, folliculardendritic cells, and B cells. In embodiments, APC targeting features maycomprise immunofeature surface(s) and/or targeting moieties that bindknown targets on APCs. In embodiments, APC targeting features maycomprise one or more B cell antigens present on a surface of syntheticnanocarriers. In embodiments, APC targeting features may also compriseone or more dimensions of the synthetic nanoparticles that is selectedto promote uptake by APCs.

In embodiments, targeting moieties for known targets on macrophages(“Mphs”) comprise any targeting moiety that specifically binds to anyentity (e.g., protein, lipid, carbohydrate, small molecule, etc.) thatis prominently expressed and/or present on macrophages (i.e.,subcapsular sinus-Mph markers). Exemplary SCS-Mph markers include, butare not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, MRP-1); CD1a (LFA-1α, α L Integrin chain); CD11b (αM Integrin chain, CR3, Mo1,C3niR, Mac-1); CD11c (αX Integrin, p150, 95, AXb2); CDw12 (p90-120);CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X,SSEA-1, 3-FAL); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X);CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb);CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a,b,cβ-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein); CD29(Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32(FCγRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV,PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin,Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5);CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP,OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1,OX-45); CD49a (VLA-1α,α1 Integrin); CD49b (VLA-2α, gpla, α2 Integrin);CD49c (VLA-3α, α3 Integrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α,α6 Integrin, gplc); CD50 (ICAM-3); CD51 (Integrin α, VNR-α,Vitronectin-Rα); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1);CD55 (DAF); CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL,P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP IIIa, β3 Integrin);CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1,gp55, NGA, LAMP-3, ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); CD65s(Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD74 (Ii,invariant chain); CD75 (sialo-masked Lactosamine); CD75S (α2,6sialylated Lactosamine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 (4F9,C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d(ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18);CD86 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor, FcαR);CD91 (α2M-R, LRP); CDw92 (p70); CDw93 (GR11); CD95 (APO-1, FAS,TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2);CD99R(CD99 Mab restricted); CD100 (SEMA4D); CD101 (IGSF2, P126, V7);CD102 (ICAM-2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); CD112 (HveB,PRR2, PVRL2, Nectin2); CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms,CSF-1R, M-CSFR); CD116 (GMCSFRα); CDw119 (IFNγR, IFNγRA); CD120a (TNFRI,p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R); CD122(IL2Rβ); CD123 (IL-3Rα); CD124 (IL-4Rα); CD127 (p90, IL-7R, IL-7Rα);CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb,CSCR2, (Tentatively renamed as CD182)); CD130 (gp130); CD131 (Common βsubunit); CD132 (Common γ chain, IL-2Rγ); CDw136 (MSP-R, RON, p158-ron);CDw137 (4-1BB, ILA); CD139; CD141 (Thrombomodulin, Fetomodulin); CD147(Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260, DEP-1); CD155 (PVR);CD156a (CD156, ADAM8, MS2); CD156b (TACE, ADAM17, cSVP); CDw156C(ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24);CD165 (AD2, gp37); CD168 (RHAMM, 1HABP, HMMR); CD169 (Sialoadhesin,Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-1α,MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1,(Formerly known as CD128a)); CD182 (CXCR2, (Formerly known as CD128b));CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195 (CCR5); CDw197(CCR7 (was CDw197)); CDw198 (CCR8); CD204 (MSR); CD205 (DEC-25); CD206(MMR); CD207 (Langerin); CDw210 (CK); CD213a (CK); CDw217 (CK); CD220(Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD226(DNAM-1, PTA1); CD230 (Prion Protein (PrP)); CD232 (VESP-R); CD244 (2B4,P38, NAIL); CD245 (p220/240); CD256 (APRIL, TALL2, TNF (ligand)superfamily, member 13); CD257 (BLYS, TALL1, TNF (ligand) superfamily,member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262(TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNBF-Rsuperfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member10d); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD277 (BT3.1, B7family: Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1, TOLL-likereceptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4, TOLL-likereceptor 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase, β3 subunit);CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e (CMRF-35L1); CD302 (DCL1);CD305 (LAIR1); CD312 (EMR2); CD315 (CD9P1); CD317 (BST2); CD321 (JAM1);CD322 (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 (gp 110,Macrosialin); and/or mannose receptor; wherein the names listed inparentheses represent alternative names.

In embodiments, targeting moieties for known targets on dendritic cells(“DCs”) comprise any targeting moiety that specifically binds to anyentity (e.g., protein, lipid, carbohydrate, small molecule, etc.) thatis prominently expressed and/or present on DCs (i.e., a DC marker).Exemplary DC markers include, but are not limited to, CD1a (R4, T6,HTA-1); CD1b (R1); CD1c (M241, R7); CD1d (R3); CD1e (R2); CD11b (αMIntegrin chain, CR3, Mol, C3niR, Mac-1); CD11c (αX Integrin, p150, 95,AXb2); CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35(CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA, T200,B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d (VLA-4α, α4Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (LFA-3); CD64 (FcγRI); CD72(Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′nucloticlase); CD74 (Ii,invariant chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15);CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1,MIR7); CD85k (ILT3, LIRS, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97(BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFRα); CD120a (TMFR1,p55); CD120b (TNFRII, p75, TNFR p80); CD123 (IL-3Rα); CD139; CD148(HPTP-η, DEP-1); CD150 (SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP); CD157(Mo5, BST-1); CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR);CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE);CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64);CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6); CD197 (CCR7 (wsCDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CD206(MMR); CD207 (Langerin); CD208 (DC-LAMP); CD209 (DCSIGN); CDw218a(IL18Rα); CDw218b (IL8Rβ); CD227 (MUC1, PUM, PEM, EMA); CD230 (PrionProtein (PrP)); CD252 (OX40L, TNF (ligand) superfamily, member 4); CD258(LIGHT, TNF (ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily, member16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276(B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3); CD283 (TLR3, TOLL-likereceptor 3); CD289 (TLR9, TOLL-like receptor 9); CD295 (LEPR); CD298(ATP1B3, Na K ATPase β3 submit); CD300a (CMRF-35H); CD300c (CMRF-35A);CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303 (BDCA2); CD304 (BDCA4);CD312 (EMR2); CD317 (BST2); CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68(gp110, Macrosialin); class II MHC; BDCA-1; Siglec-H; wherein the nameslisted in parentheses represent alternative names.

In embodiments, targeting can be accomplished by any targeting moietythat specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on B cells (i.e., B cell marker). Exemplary B cell markersinclude, but are not limited to, CD1c (M241, R7); CD1d (R3); CD2(E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9(p24, DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD11b (αMIntegrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, P150, 95,AXb2); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a, b, cβ-subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R, C3dR); CD22(BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24(BBA-1, HSA); CD25 (Tac antigen, IL-2Rα, p55); CD26 (DPP IV ectoeneyme,ADA binding protein); CD27 (T14, S152); CD29 (Platelet GPIIa, β-1integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD35 (CR1,C3b/C4b receptor); CD37 (gp52-40); CD38 (ADPribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD44 (ECMRII,H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC;CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophilin); CD47R(MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gpla, α2Integrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 Integrin);CD50 (ICAM-3); CD52 (CAMPATH-1, HES); CD53 (OX-44); CD54 (ICAM-1); CD55(DAF); CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM-1, LECAM-1,MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73(Ecto-5′-nuciotidase); CD74 (Ii, invariant chain); CD75 (sialo-maskedLactosamine); CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk antigen,BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, B29); CD80; CD81 (TAPA-1);CD82 (4F9, C33, IA4, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j(ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98 (4F2,FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD102 (ICAM-2); CD108(SEMA7A, JMH blood group antigen); CDw119 (IFNγR, IFNγRa); CD120a(TNFR1, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R);CD122 (IL2Rβ); CD124 (IL-4Rα); CD130 (gp130); CD132 (Common γ chain,IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN, M6, OX47);CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD166(ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD167a (DDR1, trkE, cak); CD171(L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, Ly64);CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197(CCR7 (was CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2); CD205(DEC-205); CDw210 (CK); CD213a (CK); CDw217 (CK); CDw218a (IL18Rα);CDw218b (IL18Rβ); CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R,IGFII-R); CD224 (GGT); CD225 (Leu13); CD226 (DNAM-1, PTA1); CD227 (MUC1,PUM, PEM, EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232(VESP-R); CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1,TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily,member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264(TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD267 (TACI, TNF-R superfamily, member 13B);CD268 (BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA, TNF-Rsuperfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3.1.B7 family:Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 subunit);CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD307 (IRTA2);CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321(JAM1); CD322 (JAM2); CDw327 (Siglec6, CD33L); CD68 (gp 100,Macrosialin); CXCR5; VLA-4; class II MHC; surface IgM; surface IgD;APRL; and/or BAFF-R; wherein the names listed in parentheses representalternative names. Examples of markers include those provided elsewhereherein.

In some embodiments, B cell targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on B cells upon activation (i.e., activated B cellmarker). Exemplary activated B cell markers include, but are not limitedto, CD1a (R4, T6, HTA-1); CD1b (R1); CD15s (Sialyl Lewis X); CD15u (3′sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1);CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80(B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BLKDD/F12); CD125 (IL-5Rα);CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152(CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253(TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1); CD289 (TLR9,TOLL-like receptor 9); and CD312 (EMR2); wherein the names listed inparentheses represent alternative names. Examples of markers includethose provided elsewhere herein.

“B cell antigen” means any antigen that naturally is or could beengineered to be recognized by a B cell, and triggers (naturally orbeing engineered as known in the art) an immune response in a B cell(e.g., an antigen that is specifically recognized by a B cell receptoron a B cell). In some embodiments, an antigen that is a T cell antigenis also a B cell antigen. In other embodiments, the T cell antigen isnot also a B cell antigen. B cell antigens include, but are not limitedto proteins, peptides, small molecules, and carbohydrates. In someembodiments, the B cell antigen is a non-protein antigen (i.e., not aprotein or peptide antigen). In some embodiments, the B cell antigen isa carbohydrate associated with an infectious agent. In some embodiments,the B cell antigen is a glycoprotein or glycopeptide associated with aninfectious agent. The infectious agent can be a bacterium, virus,fungus, protozoan, parasite or prion. In some embodiments, the B cellantigen is a poorly immunogenic antigen. In some embodiments, the B cellantigen is an abused substance or a portion thereof. In someembodiments, the B cell antigen is an addictive substance or a portionthereof. Addictive substances include, but are not limited to, nicotine,a narcotic, a cough suppressant, a tranquilizer, and a sedative. In someembodiments, the B cell antigen is a toxin, such as a toxin from achemical weapon or natural sources, or a pollutant. The B cell antigenmay also be a hazardous environmental agent. In other embodiments, the Bcell antigen is an alloantigen, an allergen, a contact sensitizer, adegenerative disease antigen, a hapten, an infectious disease antigen, acancer antigen, an atopic disease antigen, an addictive substance, axenoantigen, or a metabolic disease enzyme or enzymatic product thereof.

“Biodegradable polymer” means a polymer that degrades over time whenintroduced into the body of a subject. Biodegradable polymers, includebut are not limited to, polyesters, polycarbonates, polyketals, orpolyamides. Such polymers may comprise poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid), or polycaprolactone. In someembodiments, the biodegradable polymer comprises a block-co-polymer of apolyether, such as poly(ethylene glycol), and a polyester,polycarbonate, or polyamide or other biodegradable polymer. Inembodiments, the biodegradable polymer comprises a block-co-polymer ofpoly(ethylene glycol) and poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone. In some embodiments,however, the biodegradable polymer does not comprise a polyether, suchas poly(ethylene glycol), or consists solely of the polyether.Generally, for use as part of a synthetic nanocarrier the biodegradablepolymer is insoluble in water at pH=7.4 and at 25° C. The biodegradablepolymer, in embodiments, have a weight average molecular weight rangingfrom about 800 to about 50,000 Daltons, as determined using gelpermeation chromatography. In some embodiments, the weight averagemolecular weight is from about 800 Daltons to about 10,000 Daltons,preferably from 800 Daltons to 10,000 Daltons, as determined using gelpermeation chromatography. In other embodiments, the weight averagemolecular weight is from 1000 Daltons to 10,000 Daltons, as determinedby gel permeation chromatography. In an embodiment, the biodegradablepolymer does not comprise polyketal.

“Coadministered” means administering two or more drugs to a subject in amanner that is correlated in time. In embodiments, coadministration mayoccur through administration of two or more drugs in the same dosageform. In other embodiments, coadministration may encompassadministration of two or more drugs in different dosage forms, butwithin a specified period of time, preferably within 1 month, morepreferably within 1 week, still more preferably within 1 day, and evenmore preferably within 1 hour.

“Couple” or “Coupled” or “Couples” (and the like) means attached to orcontained within the synthetic nanocarrier. In some embodiments, thecoupling is covalent. In some embodiments, the covalent coupling ismediated by one or more linkers, polymers or a unit thereof. In someembodiments, the coupling is non-covalent. In some embodiments, thenon-covalent coupling is mediated by charge interactions, affinityinteractions, metal coordination, physical adsorption, hostguestinteractions, hydrophobic interactions, TT stacking interactions,hydrogen bonding interactions, van der Waals interactions, magneticinteractions, electrostatic interactions, dipole-dipole interactions,and/or combinations thereof. In embodiments, the coupling may arise inthe context of encapsulation within the synthetic nanocarriers, usingconventional techniques. Any of the aforementioned couplings may bearranged to be on a surface or within an inventive syntheticnanocarrier.

“Derived” means adapted or modified from the original source. Forexample, as a non-limiting example, a peptide antigen derived from aninfectious strain may have several non-natural amino acid residuessubstituted for the natural amino acid residues found in the originalantigen found in the infectious strain. The adaptations or modificationsmay be for a variety of reasons, including but not limited to increasedspecificity, easier antigen processing, or improved safety.

“Dosage form” means a drug in a medium, carrier, vehicle, or devicesuitable for administration to a subject.

“Effective amount” of an inventive composition is that amount effectivefor a certain purpose. For example, when the effective amount is for atherapeutic purpose the amount is effective for treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of a disease, disorder, and/or condition provided herein.

“Encapsulate” means to enclose within a synthetic nanocarrier,preferably enclose completely within a synthetic nanocarrier. Most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. Encapsulation isdistinct from absorbtion, which places most or all of a substance on asurface of a synthetic nanocarrier, and leaves the substance exposed tothe local environment external to the synthetic nanocarrier.

“Exhibits a pH sensitive dissociation” means that a coupling between twoentities, such as the immunomodulatory agent and the syntheticnanocarrier or immunomodulatory agent coupling moiety, is significantlyreduced or eliminated by a change in environmental pH. In embodiments,relevant pH sensitive dissociations may satisfy any of the relationshipsor combinations thereof provided herein.

“IArel(4.5)_(t)%” is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for t hours divided by the sum of theweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for t hoursplus a weight of immunomodulatory agent retained in the syntheticnanocarrier upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for t hours, expressed as weightpercent, and taken as an average across a sample of the syntheticnanocarriers. In embodiments, t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, or 30 hours. In preferred embodiments, t is 24 hours.

“IArel(7.4)_(t)%” is defined as a weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=7.4 for t hours divided by the sum of theweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for t hoursplus a weight of immunomodulatory agent retained in the syntheticnanocarrier upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=7.4 for t hours, expressed as weightpercent, and taken as an average across a sample of the syntheticnanocarriers. In embodiments, t is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, or 30 hours. In preferred embodiments, t is 24 hours.

“IA(4.5)_(t1)” is defined as a weight of immunomodulatory agent releasedupon exposure of the synthetic nanocarrier to an in vitro aqueousenvironment at pH 4.5 for t1 hours taken as an average across a sampleof the synthetic nanocarriers. “IA(4.5)_(t2)” is defined as a weight ofimmunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at pH 4.5 for t2 hourstaken as an average across a sample of the synthetic nanocarriers. t1 is4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 hours; t2 is 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 hours; and t1>t2. Inpreferred embodiments, t1 is 24 hours, and t2 is 6 hours.

“Immunomodulatory agent” means an agent that modulates an immuneresponse. “Modulate”, as used herein, refers to inducing, enhancing,stimulating, or directing an immune response. Such agents includeadjuvants that stimulate (or boost) an immune response to an antigen butis not an antigen or derived from an antigen. In some embodiments, theimmunomodulatory agent is on the surface of the synthetic nanocarrierand/or is incorporated within the synthetic nanocarrier. In embodiments,the immunomodulatory agent is coupled to the synthetic nanocarrier via apolymer or unit thereof.

In some embodiments, all of the immunomodulatory agents of a syntheticnanocarrier are identical to one another. In some embodiments, asynthetic nanocarrier comprises a number of different types ofimmunomodulatory agents. In some embodiments, a synthetic nanocarriercomprises multiple individual immunomodulatory agents, all of which areidentical to one another. In some embodiments, a synthetic nanocarriercomprises exactly one type of immunomodulatory agent. In someembodiments, a synthetic nanocarrier comprises exactly two distincttypes of immunomodulatory agents. In some embodiments, a syntheticnanocarrier comprises greater than two distinct types ofimmunomodulatory agents.

“Immunomodulatory agent coupling moiety” is any moiety through which animmunomodulatory agent is bonded to a synthetic nanocarrier. Suchmoieties include covalent bonds, such as an amide bond or ester bond, aswell as separate molecules that bond (covalently or non-covalently) theimmunomodulatory agent to the synthetic nanocarrier. Such moleculesinclude linkers or polymers or a unit thereof. For example, theimmunomodulatory agent coupling moiety can comprise a charged polymer towhich an immunomodulatory agent (e.g., an immunostimulatory nucleicacid) electrostatically bonds. As another example, the immunomodulatoryagent coupling moiety can comprise a polymer or unit thereof to whichthe immunomodulatory agent covalently bonds. In some embodiments, themoiety comprises a polyester. In other embodiments, the moiety comprisespoly(ethylene glycol), poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or a polycaprolactone. The moiety mayalso comprise a unit of any of the foregoing polymers, such as a lactideor glycolide.

“Labile immunomodulatory agent(s)” means immunomodulatory agent oragents that are unstable under physiological conditions, and degrade tothe point where they are no longer pharmacologically active. Inembodiments, labile immunomodulatory agents are observed to havesystemic half-lives of elimination of less than 24 hours, preferablyless than 12 hours, more preferably less than 10 hours, even morepreferably less than 8 hours, and still more preferably less than 6hours. In embodiments, labile immunomodulatory agents compriseimidazoquinolines, adenine derivative, or oligonucleotides that comprise5′-CG-3′, wherein C is unmethylated and wherein the oligonucleotidecomprises a backbone comprising one or more unstabilized internucleotidelinkages. In embodiments, the imidazoquinolines compriseimidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, imidazoquinoline amines, imiquimod orresiquimod.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cubic synthetic nanocarrier, the minimumdimension of a synthetic nanocarrier would be the smallest of itsheight, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 100 nm. In an embodiment, a maximum dimension ofat least 75%, preferably at least 80%, more preferably at least 90%, ofthe synthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or less than 5 μm.Preferably, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 110 nm, more preferably equal to orgreater than 120 nm, more preferably equal to or greater than 130 nm,and more preferably still equal to or greater than 150 nm. Preferably, amaximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or greater than 100 nm, more preferably equal to or greaterthan 120 nm, more preferably equal to or greater than 130 nm, morepreferably equal to or greater than 140 nm, and more preferably stillequal to or greater than 150 nm. Measurement of synthetic nanocarriersizes is obtained by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (e.g. using aBrookhaven ZetaPALS instrument).

“Obtained” means taken without adaptation or modification from theoriginal source. For example, in embodiments, antigens obtained from asource may comprise the original amino acid residue sequence found inthat source. In other embodiments, for example, antigens obtained from asource may comprise the original molecular structure found in thatsource.

“Oligonucleotide” means a nucleotide molecule having from 6 to 100nucleotides, preferably from 8 to 75 nucleotides, more preferably from10 to 50 nucleotides, still more preferably from 15 to 25 nucleotides,even still more preferably 20 nucleotides. In an embodiment according tothe invention, oligonucleotides comprise less than 100 nucleotides,preferably less than 50 nucleotides, more preferably less than 25nucleotides, and still more preferably less than 10 nucleotides. Anycytosine nucleotides (“C”) present in a 5′-CG-3′ sequence of which theoligonucleotide may be comprised are unmethylated, C present in parts ofthe oligonucleotides other than in a 5′-CG-3′ sequence of which theoligonucleotide may be comprised may be methylated, or may beunmethylated. In embodiments, inventive oligonucleotides comprise abackbone comprising one or more unstabilized internucleotide linkages(meaning internucleotide linkages that are unstable under physiologicalconditions). “Unstabilized internucleotide linkage” means a linkagebetween two nucleotides of which the oligonucleotide is comprised thatis not chemically modified to stabilize the backbone, or is chemicallymodified to destabilize the backbone of the oligonucleotide underphysiological conditions. An example of an unstablized internucleotidelinkage is a phophodiester internucleotide linkage. In embodiments, theinventive oligonucleotides' backbone comprises no stabilizing chemicalmodifications that function to stabilize the backbone underphysiological conditions. In embodiments, the inventiveoligonucleotides' backbone comprises a backbone that is not modified toincorporate phosphorothioate stabilizing chemical modifications.

“Pharmaceutically acceptable excipient” means a pharmacologicallyinactive substance added to an inventive composition to furtherfacilitate administration of the composition. Examples, withoutlimitation, of pharmaceutically acceptable excipients include calciumcarbonate, calcium phosphate, various diluents, various sugars and typesof starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

“Release Rate” means the rate that an entrapped immunomodulatory agentflows from a composition, such as a synthetic nanocarrier, into asurrounding media in an in vitro release test. First, the syntheticnanocarrier is prepared for the release testing by placing into theappropriate in vitro release media. This is generally done by exchangingthe buffer after centrifugation to pellet the synthetic nanocarrier andreconstitution of the synthetic nanocarriers using a mild condition. Theassay is started by placing the sample at 37° C. in an appropriatetemperature-controlled apparatus. A sample is removed at various timepoints.

The synthetic nanocarriers are separated from the release media bycentrifugation to pellet the synthetic nanocarriers. The release mediais assayed for the immunomodulatory agent that has dispersed from thesynthetic nanocarriers. The immunomodulatory agent is measured usingHPLC to determine the content and quality of the immunomodulatory agent.The pellet containing the remaining entrapped immunomodulatory agent isdissolved in solvents or hydrolyzed by base to free the entrappedimmunomodulatory agent from the synthetic nanocarriers. Thepellet-containing immunomodulatory agent is then also measured by HPLCto determine the content and quality of the immunomodulatory agent thathas not been released at a given time point.

The mass balance is closed between immunomodulatory agent that has beenreleased into the release media and what remains in the syntheticnanocarriers. Data are presented as the fraction released or as the netrelease presented as micrograms released over time.

“Subject” means an animal, including mammals such as humans andprimates; avians; domestic household or farm animals such as cats, dogs,sheep, goats, cattle, horses and pigs; laboratory animals such as mice,rats and guinea pigs; fish; and the like.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are expressly includedas synthetic nanocarriers.

Synthetic nanocarriers include polymeric nanoparticles. In someembodiments, synthetic nanocarriers can comprise one or more polymericmatrices. The synthetic nanocarriers, however, can also include othernanomaterials and may be, for example, lipid-polymer nanoparticles. Insome embodiments, a polymeric matrix can be surrounded by a coatinglayer (e.g., liposome, lipid monolayer, micelle, etc.). In someembodiments, the synthetic nanocarrier is not a micelle. In someembodiments, a synthetic nanocarrier may comprise a core comprising apolymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipidmonolayer, etc.). In some embodiments, the various elements of thesynthetic nanocarriers can be coupled with the polymeric matrix.

The synthetic nanocarriers may comprise one or more lipids. In someembodiments, a synthetic nanocarrier may comprise a liposome. In someembodiments, a synthetic nanocarrier may comprise a lipid bilayer. Insome embodiments, a synthetic nanocarrier may comprise a lipidmonolayer. In some embodiments, a synthetic nanocarrier may comprise amicelle. In some embodiments, a synthetic nanocarrier may comprise anon-polymeric core (e.g., metal particle, quantum dot, ceramic particle,bone particle, viral particle, proteins, nucleic acids, carbohydrates,etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer,etc.).

The synthetic nanocarriers may comprise lipid-based nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles, peptide or protein-basedparticles (such as albumin nanoparticles). Synthetic nanocarriers may bea variety of different shapes, including but not limited to spheroidal,cubic, pyramidal, oblong, cylindrical, toroidal, and the like. Syntheticnanocarriers according to the invention comprise one or more surfaces.Exemplary synthetic nanocarriers that can be adapted for use in thepractice of the present invention comprise: (1) the biodegradablenanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2)the polymeric nanoparticles of Published U.S. Patent Application20060002852 to Saltzman et al., (3) the lithographically constructednanoparticles of Published U.S. Patent Application 20090028910 toDeSimone et al., (4) the disclosure of WO 2009/051837 to von Andrian etal., or (5) the nanoparticles disclosed in Published U.S. PatentApplication 2008/0145441 to Penades et al.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersmay possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,1:5, 1:7, or greater than 1:10.

In some embodiments, synthetic nanocarriers are spheres or spheroids. Insome embodiments, synthetic nanocarriers are flat or plate-shaped. Insome embodiments, synthetic nanocarriers are cubes or cubic. In someembodiments, synthetic nanocarriers are ovals or ellipses. In someembodiments, synthetic nanocarriers are cylinders, cones, or pyramids.

It is often desirable to use a population of synthetic nanocarriers thatis relatively uniform in terms of size, shape, and/or composition sothat each synthetic nanocarrier has similar properties. For example, atleast 80%, at least 90%, or at least 95% of the synthetic nanocarriersmay have a minimum dimension or maximum dimension that falls within 5%,10%, or 20% of the average diameter or average dimension. In someembodiments, a population of synthetic nanocarriers may be heterogeneouswith respect to size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g., a polymeric core) and the shell is a secondlayer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

“T cell antigen” means any antigen that is recognized by and triggers animmune response in a T cell (e.g., an antigen that is specificallyrecognized by a T cell receptor on a T cell or an NKT cell viapresentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to aCD1 complex). In some embodiments, an antigen that is a T cell antigenis also a B cell antigen. In other embodiments, the T cell antigen isnot also a B cell antigen. T cell antigens generally are proteins orpeptides. T cell antigens may be an antigen that stimulates a CD8+ Tcell response, a CD4+ T cell response, or both. The T cell antigens,therefore, in some embodiments can effectively stimulate both types ofresponses.

In some embodiments the T cell antigen is a T-helper antigen, which is aT cell antigen that can generate an augmented response to an unrelated Bcell antigen through stimulation of T cell help. In embodiments, aT-helper antigen may comprise one or more peptides derived from tetanustoxoid, Epstein-Barr virus, influenza virus, respiratory syncytialvirus, measles virus, mumps virus, rubella virus, cytomegalovirus,adenovirus, diphtheria toxoid, or a PADRE peptide. In other embodiments,a T-helper antigen may comprise one or more lipids, or glycolipids,including but not limited to: α-galactosylceramide (α-GalCer), α-linkedglycosphingolipids (from Sphingomonas spp.), galactosyl diacylglycerols(from Borrelia burgdorferi), lypophosphoglycan (from Leishmaniadonovani), and phosphatidylinositol tetramannoside (PIM4) (fromMycobacterium leprae). For additional lipids and/or glycolipids usefulas T-helper antigens, see V. Cerundolo et al., “Harnessing invariant NKTcells in vaccination strategies.” Nature Rev Immun, 9:28-38 (2009). Inembodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cellantigen that is obtained from a source, such as a natural source. Insuch embodiments, CD4+ T-cell antigen sequences, such as those peptidesthat bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity tothe antigen obtained from the source. In embodiments, the T cellantigen, preferably a T-helper antigen, may be coupled to, or uncoupledfrom, a synthetic nanocarrier.

“Unit thereof” refers to a monomeric unit of a polymer, the polymergenerally being made up of a series of linked monomers.

“Vaccine” means a composition of matter that improves the immuneresponse to a particular pathogen or disease. A vaccine typicallycontains factors that stimulate a subject's immune system to recognize aspecific antigen as foreign and eliminate it from the subject's body. Avaccine also establishes an immunologic ‘memory’ so the antigen will bequickly recognized and responded to if a person is re-challenged.Vaccines can be prophylactic (for example to prevent future infection byany pathogen), or therapeutic (for example a vaccine against a tumorspecific antigen for the treatment of cancer). Vaccines according to theinvention may comprise one or more of the synthetic nanocarriers orcompositions provided herein.

Methods of Making the Inventive Compounds, Conjugates, or SyntheticNanocarriers

The immunomodulatory agent can be coupled to the synthetic nanocarrierin any manner such that the dissociation of the immunomodulatory agentfrom the synthetic nanocarrier satisfies the dissociation relationshipsprovided herein. Methods for determining whether or not immunomodulatoryagents of synthetic nanocarriers satisfy the dissociation relationshipsprovided herein are provided elsewhere above and in the EXAMPLES.

Oligonucleotides according to the invention may be encapsulated intosynthetic nanocarriers using a variety of methods including but notlimited to C. Astete et al., “Synthesis and characterization of PLGAnanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp.247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) andPoly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties andPossible Applications in Drug Delivery” Current Drug Delivery 1:321-333(2004); C. Reis et al., “Nanoencapsulation I. Methods for preparation ofdrug-loaded polymeric nanoparticles” Nanomedicine 2:8-21 (2006). Othermethods suitable for encapsulating oligonucleotides into syntheticnanocarriers may be used, including without limitation methods disclosedin U.S. Pat. No. 6,632,671 to Unger Oct. 14, 2003.

In some embodiments, the immunomodulatory agent is covalently coupled tothe synthetic nanocarrier via an immunomodulatory agent coupling moiety(e.g., a polymer or unit thereof). In general, a polymer or unit thereofcan be covalently coupled with an immunomodulatory agent in severalways.

The following methods or any step of the methods provided are exemplaryand may be carried out under any suitable conditions. In some cases, thereaction or any step of the methods provided may be carried out in thepresence of a solvent or a mixture of solvents. Non-limiting examples ofsolvents that may be suitable for use in the invention include, but arenot limited to, p-cresol, toluene, xylene, mesitylene, diethyl ether,glycol, petroleum ether, hexane, cyclohexane, pentane, dichloromethane(or methylene chloride), chloroform, dioxane, tetrahydrofuran (THF),dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate(EtOAc), triethylamine, acetonitrile, methyl-t-butyl ether (MTBE),N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), isopropanol (IPA),mixtures thereof, or the like. In some cases, the solvent is selectedfrom the group consisting of ethyl acetate, methylene chloride, THF,DMF, NMP, DMAC, DMSO, and toluene, or a mixture thereof.

A reaction or any step of the methods provided may be carried out at anysuitable temperature. In some cases, a reaction or any step of themethods provided is carried out at about room temperature (e.g., about25° C., about 20° C., between about 20° C. and about 25° C., or thelike). In some cases, however, the reaction or any step of the methodsprovided may be carried out at a temperature below or above roomtemperature, for example, at about −20° C., at about −10° C., at about0° C., at about 10° C., at about 30° C., about 40° C., about 50° C.,about 60° C., about 70° C., about 80° C., about 90° C., about 100° C.,about 120° C., about 140° C., about 150° C. or greater. In particularembodiments, the reaction or any step of the methods provided isconducted at temperatures between 0° C. and 120° C. In some embodiments,the reaction or any step of the methods provided may be carried out atmore than one temperature (e.g., reactants added at a first temperatureand the reaction mixture agitated at a second wherein the transitionfrom a first temperature to a second temperature may be gradual orrapid).

The reaction or any step of the methods provided may be allowed toproceed for any suitable period of time. In some cases, the reaction orany step of the methods provided is allowed to proceed for about 10minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours,about 12 hours, about 16 hours, about 24 hours, about 2 days, about 3days, about 4 days, or more. In some cases, aliquots of the reactionmixture may be removed and analyzed at an intermediate time to determinethe progress of the reaction or any step of the methods provided. Insome embodiments, a reaction or any step of the methods provided may becarried out under an inert atmosphere in anhydrous conditions (e.g.,under an atmosphere of nitrogen or argon, anhydrous solvents, etc.)

The reaction products and/or intermediates may be isolated (e.g., viadistillation, column chromatography, extraction, precipitation, etc.)and/or analyzed (e.g., gas liquid chromatography, high performanceliquid chromatography, nuclear magnetic resonance spectroscopy, etc.)using commonly known techniques. In some cases, a synthetic nanocarriermay be analyzed to determine the loading of immunomodulatory agent, forexample, using reverse phase HPLC.

The polymers may have any suitable molecular weight. For example, thepolymers may have a low or high molecular weight. Non-limiting molecularweight values include 100 Da, 200 Da, 300 Da, 500 Da, 750 Da, 1000 Da,2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da,10,000 Da, or greater. In some embodiments, the polymers have a weightaverage molecular weight of about 800 Da to about 10,000 Da. Themolecular weight of a polymer may be determined using gel permeationchromatography.

Provided below are exemplary reactions that are not intended to belimiting.

Method 1

A polymer (e.g., PLA, PLGA) or unit thereof with at least one acid endgroups is converted to a reactive acylating agent such as an acylhalide, acylimidazole, active ester, etc. using an activating reagentcommonly used in amide synthesis.

In this two-step method, the resulting activated polymer or unit thereof(e.g., PLA, PLGA) is isolated and then reacted with an immunomodulatoryagent (e.g., R848) in the presence of a base to give the desiredconjugate (e.g., PLA-R848), for example, as shown in the followingscheme:

Activating reagents that can be used to convert polymers or unitsthereof, such as PLA or PLGA, to an activated acylating form include,but are not limited to cyanuric fluoride,N,N-tetramethylfluoroformamidinium hexafluorophosphate (TFFH);Acylimidazoles, such as carbonyl diimidazole (CDI),N,N′-carbonylbis(3-methylimidazolium)triflate (CBMIT); and Activeesters, such as N-hydroxylsuccinimide (NHS or HOSu) in the presence of acarbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC),N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) orN,N′-diisopropylcarbodiimide (DIC); N,N′-disuccinimidyl carbonate (DSC);pentafluorophenol in the presence of DCC or EDC or DIC;pentafluorophenyl trifluoroacetate.

The activated polymer or unit thereof may be isolated (e.g., viaprecipitation, extraction, etc.) and/or stored under suitable conditions(e.g., at low temperature, under argon) following activation, or may beused immediately. The activated polymer or unit thereof may be reactedwith an immunomodulatory agent under any suitable conditions. In somecases, the reaction is carried out in the presence of a base and/orcatalyst. Non-limiting examples of bases/catalysts includediisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP).

Method 2

A polymer or unit thereof (e.g., PLA, PLGA having any suitable molecularweight) with an acid end group reacts with an immunomodulatory agent(e.g., R848) in the presence of an activating or coupling reagent, whichconverts the polymer or unit thereof (e.g., PLA, PLGA) to a reactiveacylating agent in situ, to give the desired conjugate (e.g., PLA-R848,PLGA-R848).

Coupling or activating agents include but are not limited to: activatingagents used in the presence of an carbodiimide such as EDC or DCC orDIC, such as 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-7-azabenzotriazole(HOAt), 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HO-Dhbt),N-Hydroxysuccinimide (NHS or HOSu), Pentafluorophenol (PFP); Activatingagents without carbodiimide: Phosphonium salts, such asO-Benzotriazol-1-yloxytris(dimethylamino) phosphoniumhexafluorophosphate (BOP),O-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP), 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP); uronium salts such asO-Benzotriazol-1-yloxytris-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and hexafluorophosphate (HBTU),O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),O-(1,2-dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyl-uroniumtetrafluoroborate (TPTU); Halouronium and halophosphonium salts such asbis(tetramethylene)fluoroformamidinium hexafluorophosphate (BTFFH),bromotris(dimethylamino) phosphonium hexafluoro-phosphate (BroP),bromotripyrrolidino phosphonium hexafluorophosphate (PyBroP) andchlorotripyrrolidino phosphonium hexafluorophosphate (PyClop);Benzotriazine derivatives such asO-(3,4-Dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TDBTU) and3-(diethyloxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT).Non-limiting examples of suitable solvents include DMF, DCM, toluene,ethyl acetate, etc., as described herein.

Method 3

Immunomodulatory agents, such as R848, can also be coupled to polymersor units thereof that are terminated in a hydroxyl group. Such polymersor units thereof include polyethylene glycol, polylactide,polylactide-co-glycolide, polycaprolactone, and other like polyesters,or units thereof. In general, the reaction proceeds as follows where animide of the general structure (IV) will react with the terminalhydroxyl of the aforementioned polymers or units thereof using acatalyst used in lactone ring opening polymerizations. The resultingreaction product (II) links the amide of the agent to the polymer orunit thereof via an ester bond. The compounds of formula (IV) and (II)are as follows:

wherein R₁═H, OH, SH, NH₂, or substituted or unsubstituted alkyl,alkoxy, alkylthio, or alkylamino; R₂═H, alkyl, or substituted alkyl; Y═Nor C; R₃ is absent if Y═N; or is H, alkyl, substituted alkyl, orcombined with R₄ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected if Y═C; R₄ is H,or substituted or unsubstituted alkyl, alkoxy, alkylthio, or alkylaminowhen not combined with R₃ to form a carbocycle or heterocycle with thecarbon atoms of the pyridine ring to which they are connected; or iscombined with R₃ to form a carbocycle or heterocycle with the carbonatoms of the pyridine ring to which they are connected; R₅ is a polymeror unit thereof; X is C, N, O, or S; R₆ and R₇ are each independently Hor substituted; and R₉, R₁₀, R₁₁, and R₁₂ are each independently H, ahalogen, OH, thio, NH₂, or substituted or unsubstituted alkyl, aryl,heterocyclic, alkoxy, aryloxy, alkylthio, arylthio, alkylamino, orarylamino.

Catalysts include, but are not limited to, phosphazine bases,1,8-diazabicycloundec-7-ene (DBU), 1,4,7-triazabicyclodecene (TBD), andN-methyl-1,4,7-triazabicyclodecene (MTDB). Other catalysts are known inthe art and provided, for example, in Kamber et al., OrganocatalyticRing-Opening Polymerization, Chem. Rev. 2007, 107, 58-13-5840.Non-limiting examples of suitable solvents include methylene chloride,chloroform, and THF.

A specific example of a reaction completed by such a method is shownhere:

wherein R₅—OH contains two hydroxyl groups (e.g., a diol, HO—R₅—OH),each of which are functionalized by reaction with an imide associatedwith R848. In some cases, HO—R₅—OH is a poly-diol such aspoly(hexamethyl carbonate)diol or polycaprolactone diol.

In embodiments where a poly-diol is employed, one of the diol groups maybe protected with a protecting group (e.g., t-butyloxycarbonyl), thusthe poly-diol would be a compound of formula HO—R₅—OP, wherein P is aprotecting group. Following reaction with an immunomodulatory agent toform a immunomodulatory agent-R₅—OP conjugate, the protecting group maybe removed and the second diol group may be reacted with any suitablereagent (e.g., PLGA, PLA).

Method 4

A conjugate (e.g., R848-PLA) can be formed via a one-pot ring-openingpolymerization of an immunomodulatory agent (e.g., R848) with a polymeror unit thereof (e.g., D/L-lactide) in the presence of a catalyst, forexample, as shown in the following scheme:

In a one-step procedure, the immunomodulatory agent and the polymer orunit thereof may be combined into a single reaction mixture comprising acatalyst. The reaction may proceed at a suitable temperature (e.g., atabout 150° C.) and the resulting conjugate may be isolated usingcommonly known techniques. Non-limiting examples of suitable catalystsinclude DMAP and tin ethylhexanoate.

Method 5

A conjugate can be formed two-step ring opening polymerization of animmunomodulatory agent (e.g., R848) with one or more polymers or unitsthereof (e.g., D/L-lactide and glycolide) in the presence of a catalyst,for example, as shown in the following scheme:

The polymers or units thereof may be first combined, and in some cases,heated (e.g., to 135° C.) to form a solution. The immunomodulatory agentmay be added to a solution comprising the polymers or units thereof,followed by addition of a catalyst (e.g., tin ethylhexanoate). Theresulting conjugate may be isolated using commonly known techniques.Non-limiting examples of suitable catalysts include DMAP and tinethylhexanoate.

In some embodiments, the immunomodulatory agent, antigen, and/ortargeting moiety can be covalently associated with a polymeric matrix.In some embodiments, covalent association is mediated by a linker. Insome embodiments, the immunomodulatory agent, antigen, and/or targetingmoiety can be noncovalently associated with a polymeric matrix. Forexample, in some embodiments, the immunomodulatory agent, antigen,and/or targeting moiety can be encapsulated within, surrounded by,and/or dispersed throughout a polymeric matrix. Alternatively oradditionally, the immunomodulatory agent, antigen, and/or targetingmoiety can be associated with a polymeric matrix by hydrophobicinteractions, charge interactions, van der Waals forces, etc.

The immunomodulatory agents can also be encapsulated within thenanocarriers. The nanocarriers, therefore, can be of any material thatis pH sensitive provided that the resulting inventive syntheticnanocarriers satisfy the dissociation relationships provided herein.Such synthetic nanocarriers are well known in the art and includepolyketal nanocarriers, pH sensitive liposomes, acid-swelling,cross-linked nanoparticles, such as those of Griset et al., J. Am. Chem.Soc. 2009, 131, 2469-2471, which in their initial state are hydrophobic,but upon cellular internalization transform to a hydrophilic structure(a hydrogel particle), and polymeric nanoparticles, such as those ofGriset, Dissertation entitled: Delivery of Paclitaxel via pH-ResponsivePolymeric Nanoparticles for Prevention of Lung Cancer and MesotheliomaRecurrence, Ohio State University, 2003. The pH sensitive syntheticnanocarriers also include those that comprise polymers that dissolve ata pH below 6 or polymers that swell at an acidic pH. In someembodiments, the synthetic nanocarriers are of a non-polyketal material.In other embodiment, the synthetic nanocarriers are not micelles.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventially. In general, a polymeric matrixcomprises one or more polymers. Polymers may be natural or unnatural(synthetic) polymers. Polymers may be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers may berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the present invention include,but are not limited to polyethylenes, polycarbonates (e.g.,poly(1,3-dioxan-2one)), polyanhydrides (e.g., poly(sebacic anhydride)),polyhydroxyacids (e.g., poly(β-hydroxyalkanoate)), polypropylfumerates,polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide),poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polyureas, polystyrenes, polyamines, and polysaccharides (e.g.,chitosan).

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g., coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with PEG, with a carbohydrate, and/or withacyclic polyacetals derived from polysaccharides (Papisov, 2001, ACSSymposium Series, 786:301).

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester),poly(ortho ester)-PEG copolymers, poly(caprolactone),poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEGcopolymers, poly(ethyleneimine), poly(ethylene imine)-PEG copolymers,poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 47-93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive compounds and synthetic nanocarriers maycomprise block copolymers, graft copolymers, blends, mixtures, and/oradducts of any of the foregoing and other polymers. Those skilled in theart will recognize that the polymers listed herein represent anexemplary, not comprehensive, list of polymers that can be of use inaccordance with the present invention.

In some embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)-400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In certain embodiments, thecarbohydrate is a sugar alcohol, including but not limited to mannitol,sorbitol, xylitol, erythritol, maltitol, and lactitol.

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing fluidic channels, spraydrying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, δ: 275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755, and also U.S. Pat. Nos.5,578,325 and 6,007,845).

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Coupling can be achieved in a variety of different ways, and can becovalent or non-covalent. Such couplings may be arranged to be on asurface or within an inventive synthetic nanocarrier. Elements of theinventive synthetic nanocarriers (such as moieties of which animmunofeature surface is comprised, targeting moieties, polymericmatrices, and the like) may be directly coupled with one another, e.g.,by one or more covalent bonds, or may be coupled by means of one or morelinkers. Additional methods of functionalizing synthetic nanocarriersmay be adapted from Published US Patent Application 2006/O002852 toSaltzman et al., Published US Patent Application 2009/O028910 toDeSimone et al., or Published International Patent ApplicationWO/2008/127532 A1 to Murthy et al.

Any suitable linker can be used in accordance with the presentinvention. Linkers may be used to form amide linkages, ester linkages,disulfide linkages, etc. Linkers may contain carbon atoms or heteroatoms(e.g., nitrogen, oxygen, sulfur, etc.). In some embodiments, a linker isan aliphatic or heteroaliphatic linker. In some embodiments, the linkeris a polyalkyl linker. In certain embodiments, the linker is a polyetherlinker. In certain embodiments, the linker is a polyethylene linker. Incertain specific embodiments, the linker is a polyethylene glycol (PEG)linker.

In some embodiments, the linker is a cleavable linker. To give but a fewexamples, cleavable linkers include protease cleavable peptide linkers,nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, pH sensitive linkers,hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers,enzyme cleavable linkers (e.g., esterase cleavable linker),ultrasound-sensitive linkers, x-ray cleavable linkers, etc. In someembodiments, the linker is not a cleavable linker.

A variety of methods can be used to couple a linker or other element ofa synthetic nanocarrier with the synthetic nanocarrier. Generalstrategies include passive adsorption (e.g., via electrostaticinteractions), multivalent chelation, high affinity non-covalent bindingbetween members of a specific binding pair, covalent bond formation,etc. (Gao et al., 2005, Curr. Op. Biotechnol., 16:63). In someembodiments, click chemistry can be used to associate a material with asynthetic nanocarrier.

Non-covalent specific binding interactions can be employed. For example,either a particle or a biomolecule can be functionalized with biotinwith the other being functionalized with streptavidin. These twomoieties specifically bind to each other noncovalently and with a highaffinity, thereby associating the particle and the biomolecule. Otherspecific binding pairs could be similarly used. Alternately,histidine-tagged biomolecules can be associated with particlesconjugated to nickel-nitrolotriaceteic acid (Ni-NTA).

For additional general information on coupling, see the journalBioconjugate Chemistry, published by the American Chemical Society,Columbus Ohio, PO Box 3337, Columbus, Ohio, 43210; “Cross-Linking,”Pierce Chemical Technical Library, available at the Pierce web site andoriginally published in the 1994-95 Pierce Catalog, and references citedtherein; Wong S S, Chemistry of Protein Conjugation and Cross-linking,CRC Press Publishers, Boca Raton, 1991; and Hermanson, G. T.,Bioconjugate Techniques, Academic Press, Inc., San Diego, 1996.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

Pharmaceutical Compositions and Methods of Use

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. In an embodiment, inventive synthetic nanocarriers are suspendedin sterile saline solution for injection together with a preservative.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting composition are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The inventive compositions may be administered by a variety of routes ofadministration, including but not limited to subcutaneous,intramuscular, intradermal, oral, parenteral, intranasal, transmucosal,rectal; ophthalmic, transdermal, transcutaneous or by a combination ofthese routes.

The compositions and methods described herein can be used to induce,enhance, stimulate, modulate, or direct an immune response. Thecompositions and methods described herein can be used in the diagnosis,prophylaxis and/or treatment of conditions such as cancers, infectiousdiseases, metabolic diseases, degenerative diseases, inflammatorydiseases, immunological diseases, or other disorders and/or conditions.The compositions and methods described herein can also be used for theprophylaxis or treatment of an addiction, such as an addiction tonicotine or a narcotic. The compositions and methods described hereincan also be used for the prophylaxis and/or treatment of a conditionresulting from the exposure to a toxin, hazardous substance,environmental toxin, or other harmful agent.

EXAMPLES Example 1 Preparation of Activated Polymer

PLA (dl-polylactide) (Resomer R202H from Boehringer-Ingelheim, KOHequivalent acid number of 0.21 mmol/g, intrinsic viscosity (iv): 0.21dl/g) (10 g, 2.1 mmol, 1.0 eq) was dissolved in dichloromethane (DCM)(35 mL). EDC (2.0 g, 10.5 mmol, 5 eq) and NHS (1.2 g, 10.5 mmol, 5 eq)were added. The solids were dissolved with the aid of sonication. Theresulting solution was stirred at room temperature for 6 days. Thesolution was concentrated to remove most of DCM and the residue wasadded to a solution of 250 mL of diethyl ether and 5 mL of MeOH toprecipitate out the activated PLA-NHS ester. The solvents were removedand the polymer was washed twice with ether (2×200 mL) and dried undervacuum to give PLA-NHS activated ester as a white foamy solid (˜8 grecovered, ¹H NMR was used to confirm the presence of NHS ester). ThePLA-NHS ester was stored under argon in a below −10 C freezer beforeuse.

Alternatively, the reaction can be performed in DMF, THF, dioxane, orCHCl3 instead of DCM. DCC can be used instead of EDC (resulting DCC-ureais filtered off before precipitation of the PLA-NHS ester from ether).The amount of EDC or DCC and NHS can be in the range of 2-10 eq of thePLA.

In the same manner, PLA with iv of 0.33 dl/g and acid number of 0.11mmol/g or PLGA (Resomer RG653H, 65% lactide-35% glycolide, iv: 0.39 dl/gand acid number 0.08 mmol/g) or PLGA (Resomer RG752H, 75% lactide-25%glycolide, iv: 0.19 dl/g and acid number of 0.22 mmol/g) is converted tothe corresponding PLA-NHS or PLGA-NHS activated ester and stored underargon in a below −10 C freezer before use.

Example 2 Preparation of Activated Polymer

PLA (R202H, acid number of 0.21 mmol/g) (2.0 g, 0.42 mmol, 1.0 eq) wasdissolved in 10 mL of dry acetonitrile. N,N′-disuccinimidyl carbonate(DSC) (215 mg, 1.26 mmol, 3.0 eq) and catalytic amount of4-(N,N-dimethylamino)pyridine (DMAP) were added. The resulting mixturewas stirred under argon for 1 day. The resulting solution wasconcentrated to almost dryness. The residue was then added to 40 mL ofether to precipitate out the polymer which was washed twice with ether(2×30 mL) and dried under vacuum to give PLA-NHS activated ester (1H NMRshowed the amount of NHS ester at about 80%).

Example 3 Preparation of Activated Polymer

PLA (R202H) (5.0 g, 1.05 mmol) was dissolved in 25 mL of anhydrous DCMand 2.5 mL of anhydrous DMF. DCC (650 mg, 3.15 mmol, 5.0 eq) andpentafluorophenol (PFP) (580 mg, 3.15 mmol, 5.0 eq) were added. Theresulting solution was stirred at room temperature for 6 days and thenconcentrated to remove DCM. The resulting residue was added to 250 mL ofether to precipitate out the activated PLA polymer which was washed withether (2×100 mL) and dried under vacuum to give PLA-PFP activated esteras a white foamy solid (4.0 g).

Example 4 Conjugation of Immunomodulatory Agent

PLA-NHS (1.0 g), R848 (132 mg, 0.42 mmol) and diisopropylethylamine(DIPEA) (0.073 mL, 0.42 mmol) were dissolved in 2 mL of dry DMF underargon. The resulting solution was heated at 50-60 C for 2 days. Thesolution was cooled to rt and added to 40 mL of de-ionized (DI) water toprecipitate out the polymer product. The polymer was then washed with DIwater (40 mL) and ether (2×40 mL) and dried at 30 C under vacuum to giveR848-PLA conjugate as a white foamy solid (0.8 g, H NMR showed theconjugation of R848 to PLA via the amide bond). The degree ofconjugation (loading) of R848 on the polymer was confirmed by HPLCanalysis as follows: a weighed amount of polymer was dissolved inTHF/MeOH and treated with 15% NaOH. The resulting hydrolyzed polymerproducts were analyzed for the amount of R848 by HPLC in comparison witha standard curve.

Example 5 Conjugation of Immunomodulatory Agent

PLA-NHS (1.0 g, 0.21 mmol, 1.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq),DIPEA (0.15 mL, 0.84 mmol, 4.0 eq) and DMAP (25 mg, 0.21 mmol, 1.0 eq)were dissolved in 2 mL of dry DMF under argon. The resulting solutionwas heated at 50-60 C for 2 days. The solution was cooled to rt andadded to 40 mL of de-ionized (DI) water to precipitate out the polymerproduct. The polymer was then washed with DI water (40 mL) and ether(2×40 mL) and dried at 30 C under vacuum to give PLA-R848 conjugate as awhite foamy solid (0.7 g, 20 mg of the polymer was hydrolyzed insolution of 0.2 mL of THF, 0.1 mL of MeOH and 0.1 mL of 15% NaOH. Theamount of R848 on the polymer was determined to be about 35 mg/g byreverse phase HPLC analysis (C18 column, mobile phase A: 0.1% TFA inwater, mobile phase B: 0.1% TFA in CH3CN, gradient).

Example 6 Conjugation of Immunomodulatory Agent

PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), DCC (260 mg, 1.26 mmol, 3.0 eq),NHS (145 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), DMAP(77 mg, 0.63 mmol, 1.5 eq) and DIPEA (0.223 mL, 1.26 mmol, 3.0 eq) weredissolved in 4 mL of dry DMF. The mixture was heated at 50-55 C for 3days. The mixture was cooled to rt and diluted with DCM. The DCC-ureawas filtered off and the filtrate was concentrated to remove DCM. Theresulting residue in DMF was added to water (40 mL) to precipitate outthe polymer product which was washed with water (40 mL), ether/DCM (40mL/4 mL) and ether (40 mL). After drying under vacuum at 30 C, thedesired PLA-R848 conjugate was obtained as a white foamy solid (1.5 g).

Example 7 Conjugation of Immunomodulatory Agent

PLA (R202H) (2.0 g, 0.42 mmol, 1.0 eq), EDC (242 mg, 1.26 mmol, 3.0 eq),HOAt (171 mg, 1.26 mmol, 3.0 eq), R848 (200 mg, 0.63 mmol, 1.5 eq), andDIPEA (0.223 mL, 1.26 mmol, 3.0 eq) were dissolved in 4 mL of dry DMF.The mixture was heated at 50-55 C for 2 days. The solution was cooled tort and added to water (40 mL) to precipitate out the polymer productwhich was washed with water (40 mL), ether/MeOH (40 mL/2 mL) and ether(40 mL). The orange colored polymer was dissolved in 4 mL of DCM and theresulting solution was added to 40 mL of ether to precipitate out thepolymer without much of the orange color. The light colored polymer waswashed with ether (40 mL). After drying under vacuum at 30 C, thedesired PLA-R848 conjugate was obtained as a light brown foamy solid(1.5 g).

Example 8 Conjugation of Immunomodulatory Agent

PLA (R202H) (1.0 g, 0.21 mmol, 1.0 eq), EDC (161 mg, 0.84 mmol, 4.0 eq),HOBt.H₂O (65 mg, 0.42 mmol, 2.0 eq), R848 (132 mg, 0.42 mmol, 2.0 eq),and DIPEA (0.150 mL, 0.84 mmol, 4.0 eq) were dissolved in 2 mL of dryDMF. The mixture was heated at 50-55° C. for 2 days. The solution wascooled to room temperature and added to water (40 mL) to precipitate outthe polymer product. The orange colored polymer was dissolved in 2 mL ofDCM and the resulting solution was added to 40 mL of ether toprecipitate out the polymer which was washed with water/acetone (40 mL/2mL) and ether (40 mL). After drying under vacuum at 30° C., the desiredPLA-R848 conjugate was obtained as an off-white foamy solid (1.0 g,loading of R848 on polymer was about 45 mg/g based on HPLC analysis andconfirmed by ¹H NMR). In the same manner, PLGA (75% Lactide)-R848 andPLGA (50% lactide)-R848 were prepared.

Example 9 Conjugation of Immunomodulatory Agent

To a round bottom flask equipped with a stir bar and condenser was addedthe imidazoquinoline, resiquimod (R-848, 218 mg, 6.93×10⁻⁴ moles), D/Llactide (1.0 g, 6.93×10⁻³ moles) and anhydrous sodium sulfate (800 mg).The flask and contents were dried under vacuum at 55° C. for 8 hours.After cooling, the flask was then flushed with argon and toluene (50 mL)was added. The reaction was stirred in an oil bath set at 120° C. untilall of the lactide had dissolved and then tin ethylhexanoate (19 mg, 15μL) was added via pipette. Heating was continued under argon for 16hours. After cooling, the reaction was diluted with ether (200 mL) andthe solution was washed with water (200 mL). The solution was dried overmagnesium sulfate, filtered and evaporated under vacuum to give 880 mg.of crude polylactic acid-R-848 conjugate. The crude polymer waschromatographed on silica using 10% methanol in methylene chloride aseluent. The fractions containing the conjugate were pooled andevaporated to give the purified conjugate. This was dried under highvacuum to provide the conjugate as a solid foam in a yield of 702 mg(57.6%). By integrating the NMR signals for the aromatic protons of thequinoline and comparing this to the integrated intensity of the lacticacid CH proton it was determined that the molecular weight of theconjugate was approximately 2 KD. GPC showed that the conjugatecontained less than 5% of free R848.

Example 10 Preparation of Low MW PLA-R848Conjugate

A solution of PLA-CO₂H (average MW: 950, DPI:1.32; 5.0 g, 5.26 mmol) andHBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at roomtemperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol)was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. for 15 h.After cooling, the mixture was diluted with EtOAc (150 mL) and washedwith 1% citric acid solution (2×40 mL), water (40 mL) and brine solution(40 mL). The solution was dried over Na₂SO₄ (10 g) and concentrated to agel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then addedand the polymer conjugate precipitated out of solution. The polymer wasthen washed with MTBE (50 mL) and dried under vacuum at room temperaturefor 2 days as a white foam (5.3 g, average MW by GPC is 1200, PDI: 1.29;R848 loading is 20% by HPLC).

Example 11 Preparation Of Low MW PLA-R848Conjugate

A solution of PLA-CO₂H (average MW: 1800, DPI:1.44; 9.5 g, 5.26 mmol)and HBTU (4.0 g, 10.5 mmol) in EtOAc (120 mL) was stirred at roomtemperature under argon for 45 min. Compound R848 (1.65 g, 5.26 mmol)was added, followed by DIPEA (5.5 mL, 31.6 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. for 15 h.After cooling, the mixture was diluted with EtOAc (150 mL) and washedwith 1% citric acid solution (2×40 mL), water (40 mL) and brine solution(40 mL). The solution was dried over Na₂SO₄ (10 g) and concentrated to agel-like residue. Methyl t-butyl ether (MTBE) (150 mL) was then addedand the polymer conjugate precipitated out of solution. The polymer wasthen washed with MTBE (50 mL) and dried under vacuum at room temperaturefor 2 days as a white foam (9.5 g, average MW by GPC is 1900, PDI: 1.53;R848 loading is 17% by HPLC).

Example 12 Conjugation of R848To PCADK Via Imide Ring Opening

The following example describes the synthesis of a polyketal, PCADK,according to a method provided in Pulendran et al, WO 2008/127532, asillustrated in step 1 below.

PCADK is synthesized in a 50 mL two-necked flask, connected to ashort-path distilling head. First, 5.5 mg of re-crystallizedp-toluenesulfonic acid (0.029 mmol, Aldrich, St. Louis, Mo.), isdissolved in 6.82 mL of ethyl acetate, and added to a 30 mL benzenesolution (kept at 10° C.), which contains 1,4-cyclohexanedimethanol(12.98 g, 90.0 mmol, Aldrich). The ethyl acetate is allowed to boil off,and distilled 2,2-dimethoxypropane (10.94 mL, 90.0 mmol, Aldrich) isadded to the benzene solution, initiating the polymerization reaction.Additional doses of 2,2-dimethoxypropane (5 mL) and benzene (25 mL) aresubsequently added to the reaction every hour for 6 hours via a meteringfunnel to compensate for 2,2-dimethoxypropane and benzene that isdistilled off. After 8 hours, the reaction is stopped by addition of 500μL of triethylamine. The polymer is isolated by precipitation in coldhexane (stored at −20° C.) followed by vacuum filtration. The molecularweight of PCADK is determined by gel permeation chromatography (GPC)(Shimadzu, Kyoto, Japan) equipped with a UV detector. THF is used as themobile phase at a flow rate of 1 ml/min. Polystyrene standards fromPolymer Laboratories (Amherst, Mass.) are used to establish a molecularweight calibration curve. This compound is used to generate the PCADKparticles in all subsequent experiments.

R848 may be conjugated to the terminal alcohol groups of the PCADKhaving molecular weight 6000 via imide ring opening, according to thestep 2 shown below.

Step 1: Preparation of PCADK

Step 2: Conjugation of PCADK to R848

In step 2, the polymer from step 1 (12 g, 2.0×10⁻³ moles) is dissolvedin methylene chloride 100 mL, and the lactam of R848 (3.3 g, 8.0×10⁻³moles) is added. This slurry is stirred as1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.835 g, 6×10⁻³ moles) isadded in a single portion. After stirring at room temperature overnight,a clear solution forms. The solution is diluted with methylene chloride(100 mL) and the solution is washed with 5% citric acid. This solutionis dried over sodium sulfate after which it is filtered and evaporatedunder vacuum. After drying under high vacuum there is obtained 11.3grams (81%) of polymer. A portion is hydrolyzed in acid and the R848content is determined to be 9% by weight.

Example 13 Conjugation of R848To Poly-Caprolactonediol Via Imide RingOpening

Imide ring opening is used to attach R854 to the terminal alcohol groupsof poly-caprolactonediol of molecular weight 2000. The polycaprolactonediol is purchased from Aldrich Chemical Company, Cat. #189421 and hasthe following structure:

The polycaprolactone diol-R854 conjugate has the following structure:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of R854 (2.4 g, 5.0×10⁻³ moles) is added. This slurryis stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³moles) is added in a single portion. After stirring at room temperaturefor 15 minutes, a clear pale yellow solution forms. The solution isdiluted with methylene chloride (100 mL) and the solution is washed with5% citric acid. This solution is dried over sodium sulfate after whichit is filtered and evaporated under vacuum. After drying under highvacuum there is obtained 5.2 grams (70%) of polymer. A portion ishydrolyzed in acid and the R848 content is determined to be 18.5% byweight.

Example 14 Conjugation Of R848To Poly-(Hexamethylene Carbonate)Diol ViaImide Ring Opening

Imide ring opening is used to attach R848 to the terminal alcohol groupsof poly-(hexamethylene carbonate)diol of molecular weight 2000. Thepoly(hexamethylene carbonate)diol is purchased from Aldrich ChemicalCompany, Cat #461164, and has the following structure:

HO—[CH₂(CH₂)₄CH₂OCO₂ ]nCH₂(CH₂)₄CH₂—OH.

The poly(hexamethylene carbonate)diol-R848 conjugate has the followingstructure:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of R848 (2.06 g, 5.0×10⁻³ moles) is added. This slurryis stirred as 1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³moles) is added in a single portion. After stirring at room temperatureovernight a clear pale yellow solution forms. The solution is dilutedwith methylene chloride (100 mL) and the solution is washed with 5%citric acid. This solution is dried over sodium sulfate after which itis filtered and evaporated under vacuum. After drying under high vacuumthere is obtained 5.9 grams (84%) of polymer. NMR is used to determinethe R848 content which is determined to be 21%.

Example 15 Polylactic Acid Conjugates of an Imidazoquinoline Using a TinEthylhexanoate Catalyst

To a two necked round bottom flask equipped with a stir bar andcondenser was added the imidazoquinoline resiquimod (R-848, 100 mg,3.18×10⁻⁴ moles), D/L lactide (5.6 g, 3.89×10⁻² moles) and anhydroussodium sulfate (4.0 g). The flask and contents were dried under vacuumat 50° C. for 8 hours. The flask was then flushed with argon and toluene(100 mL) was added. The reaction was stirred in an oil bath set at 120°C. until all of the lactide had dissolved and then tin ethylhexanoate(75 mg, 60 L) was added via pipette. Heating was continued under argonfor 16 hours. After cooling, water (20 mL) was added and stirring wascontinued for 30 minutes. The reaction was diluted with additionaltoluene (200 mL) and was then washed with water (200 mL). The toluenesolution was then washed in turn with 10% sodium chloride solutioncontaining 5% conc. Hydrochloric acid (200 mL) followed by saturatedsodium bicarbonate (200 mL). TLC (silica, 10% methanol in methylenechloride) showed that the solution contained no free R-848. The solutionwas dried over magnesium sulfate, filtered and evaporated under vacuumto give 3.59 grams of polylactic acid-R-848 conjugate. A portion of thepolymer was hydrolyzed in base and examined by HPLC for R-848 content.By comparison to a standard curve of R-848 concentration vs. HPLCresponse, it was determined that the polymer contained 4.51 mg of R-848per gram of polymer. The molecular weight of the polymer was determinedby GPC to be about 19,000.

Example 16 Low Molecular Weight Polylactic Acid Conjugates of anImidazoquinoline

To a round bottom flask equipped with a stir bar and condenser was addedthe imidazoquinoline, resiquimod (R-848, 218 mg, 6.93×10⁻⁴ moles), D/Llactide (1.0 g, 6.93×10⁻³ moles) and anhydrous sodium sulfate (800 mg).The flask and contents were dried under vacuum at 55° C. for 8 hours.After cooling, the flask was then flushed with argon and toluene (50 mL)was added. The reaction was stirred in an oil bath set at 120° C. untilall of the lactide had dissolved and then tin ethylhexanoate (19 mg, 15μL) was added via pipette. Heating was continued under argon for 16hours. After cooling, the reaction was diluted with ether (200 mL) andthe solution was washed with water (200 mL). The solution was dried overmagnesium sulfate, filtered and evaporated under vacuum to give 880 mg.of crude polylactic acid-R-848 conjugate. The crude polymer waschromatographed on silica using 10% methanol in methylene chloride aseluent. The fractions containing the conjugate were pooled andevaporated to give the purified conjugate. This was dried under highvacuum to provide the conjugate as a solid foam in a yield of 702 mg(57.6%). By integrating the NMR signals for the aromatic protons of thequinoline and comparing this to the integrated intensity of the lacticacid CH proton it was determined that the molecular weight of theconjugate was approximately 2 KD. GPC showed that the conjugatecontained less than 5% of free R848.

Example 17 Low Molecular Weight Polylactic Acid Co-Glycolic AcidConjugates of an Imidazoquinoline

To a round bottom flask equipped with a stir bar and condenser was addedthe imidazoquinoline, resiquimod (R-848, 436 mg, 1.39×10⁻³ moles),glycolide (402 mg, 3.46×10⁻³ moles), D/L lactide (2.0 g, 1.39×10⁻²moles) and anhydrous sodium sulfate (1.6 g). The flask and contents weredried under vacuum at 55° C. for 8 hours. After cooling, the flask wasthen flushed with argon and toluene (60 mL) was added. The reaction wasstirred in an oil bath set at 120° C. until all of the R848, glycolideand lactide had dissolved and then tin ethylhexanoate (50 mg, 39 μL) wasadded via pipette. Heating was continued under argon for 16 hours. Aftercooling, the reaction was diluted with ethyl acetate (200 mL) and thesolution was washed with water (200 mL). The solution was dried overmagnesium sulfate, filtered and evaporated under vacuum to give crudePLGA-R-848 conjugate. The crude polymer was chromatographed on silicausing 10% methanol in methylene chloride as eluent. The fractionscontaining the conjugate were pooled and evaporated to give the purifiedconjugate. This was dried under high vacuum to provide the conjugate asa solid foam in a yield of 1.55 g (54.6%). By integrating the NMRsignals for the aromatic protons of the quinoline and comparing this tothe integrated intensity of the lactic acid CH proton it was determinedthat the molecular weight of the conjugate was approximately 2 KD. GPCshowed that the conjugate contained no detectable free R848.

Example 18 Polylactic Acid Conjugates of an Imidazoquinoline Using aLithium Diisopropylamide Catalysis

The imidazoquinoline (R-848), D/L lactide, and associated glassware wereall dried under vacuum at 50° C. for 8 hours prior to use. To a roundbottom flask equipped with a stir bar and condenser was added the R-848(33 mg, 1.05×10⁻⁴ moles), and dry toluene (5 mL). This was heated toreflux to dissolve all of the R-848. The solution was stirred undernitrogen and cooled to room temperature to provide a suspension offinely divided R-848. To this suspension was added a solution of lithiumdiisopropyl amide (2.0 M in THF, 50 μL, 1.0×10⁻⁴ moles) after whichstirring was continued at room temperature for 5 minutes. The paleyellow solution that had formed was added via syringe to a hot (120° C.)solution of D/L lactide (1.87 g, 1.3×10⁻² moles) under nitrogen. Theheat was removed and the pale yellow solution was stirred at roomtemperature for one hour. The solution was diluted with methylenechloride (200 mL) and this was then washed with 1% hydrochloric acid(2×50 mL) followed by saturated sodium bicarbonate solution (50 mL). Thesolution was dried over magnesium sulfate, filtered and evaporated undervacuum to give the polylactic acid-R-848 conjugate. TLC (silica, 10%methanol in methylene chloride) showed that the solution contained nofree R-848. The polymer was dissolved in methylene chloride (10 mL) andthe solution was dripped into stirred hexane (200 mL). The precipitatedpolymer was isolated by decantation and was dried under vacuum to give1.47 grams of the polylactic acid—R-848 conjugate as a white solid. Aportion of the polymer was hydrolyzed in base and examined by HPLC forR-848 content. By comparison to a standard curve of R-848 concentrationvs. HPLC response, it was determined that the polymer contained 10.96 mgof R-848 per gram of polymer.

Example 19 Attachment of Immunomodulatory Agent to Low MW PLA

PLA (D/L-polylactide) with MW of 5000 (10.5 g, 2.1 mmol, 1.0 eq) isdissolved in dichloromethane (DCM) (35 mL). EDC (2.0 g, 10.5 mmol, 5 eq)and NHS (1.2 g, 10.5 mmol, 5 eq) are added. The resulting solution isstirred at room temperature for 3 days. The solution is concentrated toremove most of DCM and the residue is added to a solution of 250 mL ofdiethyl ether and 5 mL of MeOH to precipitate out the activated PLA-NHSester. The solvents are removed and the polymer is washed twice withether (2×200 mL) and dried under vacuum to give PLA-NHS activated esteras a white foamy solid (˜8 g recovered, H NMR can be used to confirm thepresence of NHS ester). The PLA-NHS ester is stored under argon in abelow −10° C. freezer before use.

Alternatively, the reaction can be performed in DMF, THF, dioxane, orCHCl₃ instead of DCM. DCC can be used instead of EDC (resulting DCC-ureais filtered off before precipitation of the PLA-NHS ester from ether).The amount of EDC or DCC and NHS can be in the range of 2-10 eq of thePLA.

Example 20 Attachment of Immunomodulatory Agent to Low MW PLGA

In the same manner as provided above for polymer activation, low MW PLGAwith 50% to 75% glycolide is converted to the corresponding PLGA-NHSactivated ester and is stored under argon in a below −10° C. freezerbefore use.

Example 21 One-Pot Ring-Opening Polymerization of R848 with D/L-Lactidein the Presence of a Catalyst

A mixture of R848 (0.2 mmol, 63 mg), D/L-lactide (40 mmol, 5.8 g), and4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol) in 2 mL of anhydroustoluene was heated slowly to 150° C. (oil bath temperature) andmaintained at this temperature for 18 h (after 3 hr, no R848 was left).The mixture was cooled to ambient temperature and the resulting mixturewas quenched with water (50 mL) to precipitate out the resultingpolymer, R848-PLA. The polymer was then washed sequentially with 45 mLeach of MeOH, iPrOH, and ethyl ether. The polymer was dried under vacuumat 30° C. to give an off-white puffy solid (5.0 g). Polymeric structurewas confirmed by ¹H NMR in CDCl₃. A small sample of the polymer wastreated with 2 N NaOH aq in THF/MeOH to determine the loading of R848 onthe polymer by reverse phase HPLC. The loading of R848 is 3 mg per gramof polymer (0.3% loading-27.5% of theory).

Example 22 Two Step Ring Opening Polymerization of R848 with D/L-Lactideand Glycolide

A mixture of D/L-lactide (10.8 g, 0.075 moles) and glycolide (2.9 g,0.025 moles) was heated to 135° C. under argon. Once all of thematerials had melted and a clear solution had resulted, R848 (1.08 g,3.43×10⁻³ moles) was added. This solution was stirred at 135° C. under aslow stream of argon for one hour. Tin ethylhexanoate (150 μL) was addedand heating was continued for 4 hours. After cooling, the solid palebrown mass was dissolved in methylene chloride (250 mL) and the solutionwas washed with 5% tartaric acid solution (2×200 mL). The methylenechloride solution was dried over magnesium sulfate, filtered, and thenconcentrated under vacuum. The residue was dissolved in methylenechloride (20 mL) and 2-propanol (250 mL) was added with stirring. Thepolymer that separated was isolated by decantation of the 2-propanol andwas dried under high vacuum. NMR showed that the polymer was 71.4%lactide and 28.6% glycolide with a molecular weight of 4000. The loadingof R848 was close to theoretical by NMR.

Example 23 Preparation of PLGA-R848Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) in anhydrous EtOAc(160 mL) was stirred at room temperature under argon for 50 minutes.Compound R848 (2.2 g, 7 mmol) was added, followed bydiisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture was stirredat room temperature for 6 h and then at 50-55° C. overnight (about 16h). After cooling, the mixture was diluted with EtOAc (200 mL) andwashed with saturated NH₄Cl solution (2×40 mL), water (40 mL) and brinesolution (40 mL). The solution was dried over Na₂SO₄ (20 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) wasthen added and the polymer conjugate precipitated out of solution. Thepolymer was then washed with IPA (4×50 mL) to remove residual reagentsand dried under vacuum at 35-40° C. for 3 days as a white powder (10.26g, MW by GPC is 5200, R848 loading is 12% by HPLC).

Example 24 Preparation of PLGA-854A Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (20 mL) was stirred at room temperature under argon for 45minutes. Compound 845A (0.29 g, 0.7 mmol) was added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture wasstirred at room temperature for 6 h and then at 50-55° C. overnight(about 15 h). After cooling, the mixture was diluted with EtOAc (100 mL)and washed with saturated NH4Cl solution (2×20 mL), water (20 mL) andbrine solution (20 mL). The solution was dried over Na₂SO₄ (10 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (40 mL) wasthen added and the polymer conjugate precipitated out of solution. Thepolymer was then washed with IPA (4×25 mL) to remove residual reagentsand dried under vacuum at 35-40° C. for 2 days as a white powder (1.21g, MW by GPC is 4900, 854A loading is 14% by HPLC).

Example 25 Preparation of PLGA-BBHA Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) was stirred at room temperature under argon for 30minutes. Compound BBHA (0.22 g, 0.7 mmol) in 2 mL of dry DMSO was added,followed by diisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). Themixture was stirred at room temperature for 20 h. Additional amounts ofHBTU (0.53 g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) were added and themixture was heated at 50-55° C. for 4 h. After cooling, the mixture wasdiluted with EtOAc (100 mL) and washed with saturated NH4Cl solution 20mL), water (2×20 mL) and brine solution (20 mL). The solution was driedover Na₂SO₄ (10 g) and concentrated to a gel-like residue. Isopropylalcohol (IPA) (35 mL) was then added and the brownish polymer conjugateprecipitated out of solution. The polymer was then washed with IPA (2×20mL) to remove residual reagents and dried under vacuum at 35-40° C. for2 days as a brownish powder (1.1 g).

Example 26 Conjugation of R848 to Polyglycine, a Polyamide

The t-butyloxycarbonyl (tBOC) protected polyglycine carboxylic acid (I)is prepared by ring opening polymerization of glycine N-carboxyanhydride(Aldrich cat #369772) using 6-aminohexanoic acid benzyl ester (Aldrichcat #S33465) by the method of Aliferis et al. (Biomacromolecules, 5,1653, (2004)). Protection of the end amino group as the t-BOC carbamatefollowed by hydrogenation over palladium on carbon to remove the benzylester completes the synthesis of BOC protected polyglycine carboxylicacid (I).

A mixture of BOC-protected polyglycine carboxylic acid (5 gm, MW=2000,2.5×10⁻³ moles) and HBTU (3.79 gm, 1.0×10⁻² moles) in anhydrous DMF (100mL) is stirred at room temperature under argon for 50 minutes. Then R848(1.6 gm, 5.0×10⁻³ moles) is added, followed by diisopropylethylamine (4mL, 2.2×10⁻² moles). The mixture is stirred at RT for 6 h and then at50-55° C. overnight (16 h). After cooling, the DMF is evaporated undervacuum and the residue is triturated in EtOAc (100 mL). The polymer isisolated by filtration and the polymer is then washed with 2-propanol(4×25 mL) to remove residual reagents and dried under vacuum at 35-40°C. for 3 days. The polymer is isolated as an off white solid in a yieldof 5.1 g (88%). The R848 loading can be determined by NMR is 10.1%.

The t-BOC protecting group is removed using trifluoroacetic acid and theresulting polymer is grafted to PLA with carboxyl end groups byconventional methods.

Example 27 Preparation of a PLGA Conjugate of thePolyglycine/R848Polymer

Step 1: A t-BOC protected polyglycine/R848 conjugate (5 g) is dissolvedin trifluoroacetic acid (25 mL) and this solution is warmed at 50° C.for one hour. After cooling, the trifluoroacetic acid is removed undervacuum and the residue is triturated in ethyl acetate (25 mL). Thepolymer is isolated by filtration and is washed well with 2-propanol.After drying under vacuum there is obtained 4.5 grams of polymer as anoff white solid.

Step 2: A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A,acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) inanhydrous DMF (100 mL) is stirred at RT under argon for 50 minutes. Thepolymer from above (1.4 g, 7 mmol) dissolved in dry DMF (20 mL) isadded, followed by diisopropylethylamine (DIPEA) (5 mL, 28 mmol). Themixture is stirred at RT for 6 h and then at 50-55° C. overnight (16 h).After cooling, the DMF is evaporated under vacuum, and the residue isdissolved in methylene chloride (50 mL). The polymer is precipitated bythe addition of 2-propanol (200 mL). The polymer is isolated bydecantation and is washed with 2-propanol (4×50 mL) to remove residualreagents and then dried under vacuum at 35-40 C overnight. There isobtained 9.8 g (86%) of the block copolymer.

Example 28 Preparation of PLGA-2-Butoxy-8-Hydroxy-9-Benzyl AdenineConjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (I)(0.22 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture isstirred at room temperature for 20 h. Additional amounts of HBTU (0.53g, 1.4 mmol) and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture isheated at 50-55° C. for 4 h. After cooling, the mixture is diluted withEtOAc (100 mL) and washed with saturated NH₄Cl solution 20 mL), water(2×20 mL) and brine solution (20 mL). The solution is dried over Na₂SO₄(10 g) and concentrated to a gel-like residue. Isopropyl alcohol (IPA)(35 mL) is then added and the brownish polymer conjugate precipitatesout of solution. The polymer is then washed with IPA (2×20 mL) to removeresidual reagents and dried under vacuum at 35-40° C. for 2 days as abrownish powder (1.0 g).

Example 29 Preparation Of PLGA-2,9-Dibenzyl-8-Hydroxyadenine Conjugate

A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A, acid number0.7 mmol/g, 1.0 g, 7.0 mmol) and HBTU (0.8 g, 2.1 mmol) in anhydrousEtOAc (30 mL) is stirred at RT under argon for 30 minutes. Compound (II)(0.24 g, 0.7 mmol) in 2 mL of dry DMSO is added, followed bydiisopropylethylamine (DIPEA) (0.73 mL, 4.2 mmol). The mixture isstirred at RT for 20 h. Additional amounts of HBTU (0.53 g, 1.4 mmol)and DIPEA (0.5 mL, 2.8 mmol) are added and the mixture is heated at50-55° C. for 4 h. After cooling, the mixture is diluted with EtOAc (100mL) and washed with saturated NH₄Cl solution 20 mL), water (2×20 mL) andbrine solution (20 mL). The solution is dried over Na₂SO₄ (10 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (35 mL) isthen added and the brownish polymer conjugate precipitated out ofsolution. The polymer is then washed with IPA (2×20 mL) to removeresidual reagents and dried under vacuum at 35-40° C. for 2 days as abrownish powder (1.2 g).

Example 30 Imide Ring Opening Used To Attach2-Pentyl-8-Hydroxy-9-Benzyladenine to the Terminal Alcohol Groups ofPoly-Hexamethylene Carbonate)Diol of Molecular Weight 2000

The poly(hexamethylene carbonate)diol is purchased from Aldrich ChemicalCompany, Cat #461164.

Poly(hexamethylene carbonate)diol:

HO—[CH₂(CH₂)₄CH₂OCO₂ ]nCH₂(CH₂)₄—CH₂—OH

Poly(hexamethylene carbonate)diol-8-oxoadenine conjugate:

The polymer (5 g, 2.5×10⁻³ moles) is dissolved in methylene chloride 25mL and the lactam of 2-pentyl-8-hydroxy-9-benzyladenine (2.05 g,5.0×10⁻³ moles) is added. This slurry is stirred as1,5,7-triazabicyclo-[4,4,0]dec-5-ene (TBD, 0.557 g, 4×10⁻³ moles) isadded in a single portion. After stirring at room temperature overnighta clear pale yellow solution forms. The solution is diluted withmethylene chloride (100 mL), and the solution is washed with 5% citricacid. This solution is dried over sodium sulfate after which it isfiltered and evaporated under vacuum. After drying under high vacuumthere is obtained 5.5 grams (78%) of polymer. NMR is used to determinethe benzyladenine content which is 18%.

Example 31 Nicotine-PEG-PLA Conjugates

A 3-nicotine-PEG-PLA polymer was synthesized as follows:

First, monoamino poly(ethylene glycol) from JenKem® with a molecularweight of 3.5 KD (0.20 gm, 5.7×10-5 moles) and an excess of4-carboxycotinine (0.126 gm, 5.7×10-4 moles) were dissolved indimethylformamide (5.0 mL). The solution was stirred anddicyclohexylcarbodiimide (0.124 gm, 6.0×10-4 moles) was added. Thissolution was stirred overnight at room temperature. Water (0.10 mL) wasadded and stirring was continued for an additional 15 minutes. Theprecipitate of dicyclohexylurea was removed by filtration and thefiltrates were evaporated under vacuum. The residue was dissolved inmethylene chloride (4.0 mL) and this solution was added to diethyl ether(100 mL). The solution was cooled in the refrigerator for 2 hours andthe precipitated polymer was isolated by filtration. After washing withdiethyl ether, the solid white polymer was dried under high vacuum. Theyield was 0.188 gm. This polymer was used without further purificationfor the next step.

The cotinine/PEG polymer (0.20 gm, 5.7×10-5 moles) was dissolved in drytetrahydrofuran (10 mL) under nitrogen and the solution was stirred as asolution of lithium aluminum hydride in tetrahydrofuran (1.43 mL of2.0M, 2.85×10-3 moles) was added. The addition of the lithium aluminumhydride caused the polymer to precipitate as a gelatinous mass. Thereaction was heated to 80° C. under a slow stream of nitrogen and thetetrahydrofuran was allowed to evaporate. The residue was then heated at80° C. for 2 hours. After cooling, water (0.5 mL) was cautiously added.Once the hydrogen evolution had stopped, 10% methanol in methylenechloride (50 mL) was added and the reaction mixture was stirred untilthe polymer had dissolved. This mixture was filtered through Celite®brand diatomaceous earth (available from EMD Inc. as Celite® 545, part#CX0574-3) and the filtrates were evaporated to dryness under vacuum.The residue was dissolved in methylene chloride (4.0 mL) and thissolution was slowly added to diethyl ether (100 mL). The polymerseparated as a white flocculent solid and was isolated bycentrifugation. After washing with diethyl ether, the solid was driedunder vacuum. The yield was 0.129 gm.

Next, a 100 mL round bottom flask, equipped with a stir bar and refluxcondenser was charged with the PEG/nicotine polymer (0.081 gm, 2.2×10-5moles), D/L lactide (0.410 gm, 2.85×10-3 moles) and anhydrous sodiumsulfate (0.380 gm). This was dried under vacuum at 55° C. for 8 hours.The flask was cooled and flushed with argon and then dry toluene (10 mL)was added. The flask was placed in an oil bath set at 120° C., and oncethe lactide had dissolved, tin ethylhexanoate (5.5 mg, 1.36×10-5 moles)was added. The reaction was allowed to proceed at 120° C. for 16 hours.After cooling to room temperature, water (15 mL) was added and stirringwas continued for 30 minutes. Methylene chloride (200 mL) was added, andafter agitation in a separatory funnel, the phases were allowed tosettle. The methylene chloride layer was isolated and dried overanhydrous magnesium sulfate. After filtration to remove the dryingagent, the filtrates were evaporated under vacuum to give the polymer asa colorless foam. The polymer was dissolved in tetrahydrofuran (10 mL)and this solution was slowly added to water (150 mL) with stirring. Theprecipitated polymer was isolated by centrifugation and the solid wasdissolved in methylene chloride (10 mL). The methylene chloride wasremoved under vacuum and the residue was dried under vacuum.3-nicotine-PEG-PLA polymer yield was 0.38 gm.

Example 32 Synthetic Nanocarrier Formulation

For encapsulated adjuvant formulations, Resiquimod (aka R848) wassynthesized according to the synthesis provided in Example 99 of U.S.Pat. No. 5,389,640 to Gerster et al.

R848 was conjugated to PLA by a method provided above, and the PLAstructure was confirmed by NMR.

PLA-PEG-nicotine conjugate was prepared according to Example 31.

PLA was purchased (Boehringer Ingelheim Chemicals, Inc., 2820 NorthNormandy Drive, Petersburg, Va. 23805). The polyvinyl alcohol (Mw=11KD-31 KD, 85-89% hydrolyzed) was purchased from VWR scientific.Ovalbumin peptide 323-339 was obtained from Bachem Americas Inc. (3132Kashiwa Street, Torrance Calif. 90505. Part #4064565).

The above materials were used to prepare the following solutions:

-   -   1. Resiquimod (R848)@10 mg/mL and PLA@100 mg/mL in methylene        chloride or PLA-R848 conjugate@100 mg/mL in methylene chloride    -   2. PLA-PEG-nicotine in methylene chloride@100 mg/mL    -   3. PLA in methylene chloride@100 mg/mL    -   4. Ovalbumin peptide 323-339 in water@10 or 69 mg/mL    -   5. Polyvinyl alcohol in water@50 mg/mL.

Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL), solution #3 (0.25to 0.5 mL) and solution #4 (0.1 mL) were combined in a small vial andthe mixture was sonicated at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. To this emulsion was added solution #5(2.0 mL) and sonication at 35% amplitude for 40 seconds using theBranson Digital Sonifier 250 forms the second emulsion. This was addedto a beaker containing phosphate buffer solution (30 mL) and thismixture was stirred at room temperature for 2 hours to form thenanoparticles.

To wash the particles a portion of the nanoparticle dispersion (7.4 mL)was transferred to a centrifuge tube and spun at 5,300 g for one hour,supernatant was removed, and the pellet was re-suspended in 7.4 mL ofphosphate buffered saline. The centrifuge procedure was repeated and thepellet was re-suspended in 2.2 mL of phosphate buffered saline for afinal nanoparticle dispersion of about 10 mg/mL.

Example 33 Double Emulsion with Multiple Primary Emulsions Materials

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T cellepitope of Ovalbumin protein, was purchased from Bachem Americas Inc.(3132 Kashiwa Street, Torrance Calif. 90505.)

Resiquimod (aka R848) was synthesized according to a method provided inU.S. Pat. No. 6,608,201.

PLA-R848, resiquimod, was conjugated to PLA with a molecular weight ofapproximately 2,500 Da according to a method provided above.

PLGA-R848, resiquimod, was conjugated to PLGA with a molecular weight ofapproximately 4,100 Da according to a method provided above.

PS-1826 DNA oligonucleotide with fully phosphorothioated backbone havingnucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodiumcounter-ion was purchased from Oligos Etc (9775 SW Commerce Circle C-6,Wilsonville, Oreg. 97070.)

PO-1826 DNA oligonucleotide with phosphodiester backbone havingnucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′ with a sodiumcounter-ion was purchased from Oligos Etc. (9775 SW Commerce Circle C-6,Wilsonville, Oreg. 97070.)

PLA with an inherent viscosity of 0.21 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. ProductCode 100 □L 2A.)

PLA with an inherent viscosity of 0.71 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. ProductCode 100 □L 7A.)

PLA with an inherent viscosity of 0.19 dL/g was purchased fromBoehringer Ingelheim Chemicals, Inc. (Petersburg, Va. Product CodeR202H.)

PLA-PEG-nicotine with a molecular weight of approximately 18,500 to22,000 Da was prepared according to a method provided above.

PLA-PEG-R848 with a molecular weight of approximately 15,000 Da wassynthesized was prepared according to a method provided above.

Polyvinyl alcohol (Mw=11,000-31,000, 87-89% hydrolyzed) was purchasedfrom J.T. Baker (Part Number U232-08).

Batches were produced using a double emulsion process with multipleprimary emulsions. The table below references the solution suffix (e.g.,B in Solution #1 column indicates Solution #1B was used) and volume ofsolution used.

Solution Solution Solution Solution Solution Sample #1 #2 #3 #4 #5Number (Volume) (Volume) (Volume) (Volume) (Volume) 1 B (0.1 ml) C (1.0ml) A (0.1 ml) C (1.0 ml) A (2.0 ml) 2 A (0.2 ml) A (1.0 ml) A (0.1 ml)A (1.0 ml) A (3.0 ml) 3 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A(3.0 ml) 4 A (0.2 ml) B (1.0 ml) A (0.1 ml) B (1.0 ml) A (3.0 ml)Solution 1A: Ovalbumin peptide 323-339 @ 35 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.Solution 1B: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.Solution 2A: 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml inmethylene chloride. The solution was prepared by first preparing twoseparate solutions at room temperature: 0.21-IV PLA @ 100 mg/mL in puremethylene chloride and PLA-PEG-nicotine @ 100 mg/mL in pure methylenechloride. The final solution was prepared by adding 3 parts PLA solutionfor each part of PLA-PEG-nicotine solution. Solution 2B: 0.71-IV PLA @75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml in methylene chloride. Thesolution was prepared by first preparing two separate solutions at roomtemperature: 0.71-IV PLA @ 100 mg/mL in pure methylene chloride andPLA-PEG-nicotine @ 100 mg/mL in pure methylene chloride. The finalsolution was prepared by adding 3 parts PLA solution for each part ofPLA-PEG-nicotine solution. Solution 2C: 0.19-IV PLA @ 75 mg/mL andPLA-PEG-nicotine @ 25 mg/ml in methylene chloride. The solution wasprepared by first preparing two separate solutions at room temperature:0.19-IV PLA @ 100 mg/mL in pure methylene chloride and PLA-PEG-nicotine@ 100 mg/mL in pure methylene chloride. The final solution was preparedby adding 3 parts PLA solution for each part of PLA-PEG-nicotinesolution. Solution 3A: Oligonucleotide (either PS-1826 or PO-1826) @ 200mg/ml in purified water. The solution was prepared by dissolvingoligonucleotide in purified water at room temperature. Solution 4A: Sameas Solution #2A. Solution 4B: Same as Solution #2B. Solution 4C: Same asSolution #2C. Solution 5A: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8phosphate buffer.

Two separate primary water in oil emulsions were prepared. W1/O2 wasprepared by combining solution 1 and solution 2 in a small pressure tubeand sonicating at 50% amplitude for 40 seconds using a Branson DigitalSonifier 250. W3/O4 was prepared by combining solution 3 and solution 4in a small pressure tube and sonicating at 50% amplitude for 40 secondsusing a Branson Digital Sonifier 250. A third emulsion with two inneremulsion ([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.5 ml ofeach primary emulsion (W1/O2 and W3/O4) and solution 5 and sonicating at30% amplitude for 40 to 60 seconds using the Branson Digital Sonifier250.

The third emulsion was added to a beaker containing 70 mM phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow for the methylene chloride to evaporate and for the nanocarriersto form. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and spinning at 13,823 g forone hour, removing the supernatant, and re-suspending the pellet inphosphate buffered saline. The washing procedure was repeated and thepellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

The amounts of oligonucleotide and peptide in the nanocarrier weredetermined by HPLC analysis.

Example 34 Standard Double Emulsion Materials

As provided in Example 33 above.

Batches were produced using a standard double emulsion process. Thetable below references the solution suffix (e.g., B in Solution #1column indicates Solution #1B was used) and volume of solution used.

Solution Solution Solution Sample #1 #2 Solution #3 Solution #4 #5Number (Volume) (Volume) (Volume) (Volume) (Volume) 1 A (0.1 ml) A A(0.25 ml) None A (2.0 ml) (0.75 ml) 2 A (0.1 ml) None A (0.25 ml) A(0.75 ml) A (2.0 ml) 3 A (0.1 ml) B A (0.25 ml) None A (2.0 ml) (0.75ml) 4 B (0.1 ml) C A (0.25 ml) None B (2.0 ml) (0.75 ml) 5 B (0.1 ml) DA (0.25 ml) A (0.50 ml) B (2.0 ml) (0.25 ml) 6 C (0.2 ml) None A (0.25ml) A (0.75 ml) B (2.0 ml) 7 D (0.1 ml) None A (0.25 ml) A (0.75 ml) B(2.0 ml) Solution 1A: Ovalbumin peptide 323-339 @ 69 mg/mL in de-ionizedwater. The solution was prepared by slowly adding ovalbumin peptide tothe water while mixing at room temperature. Solution 1B: Ovalbuminpeptide 323-339 @ 70 mg/mL in dilute hydrochloric acid aqueous solution.The solution was prepared by dissolving ovalbumin peptide in 0.13Nhydrochloric acid solution at room temperature. Solution 1C:Oligonucleotide (PS-1826) @ 50 mg/ml in purified water. The solution wasprepared by dissolving oligonucleotide in purified water at roomtemperature. Solution 1D: Ovalbumin peptide 323-339 @ 17.5 mg/mL indilute hydrochloric acid aqueous solution. The solution was prepared bydissolving ovalbumin peptide @ 70 mg/ml in 0.13N hydrochloric acidsolution at room temperature and then diluting the solution with 3 partspurified water per one part of starting solution. Solution 2A: R848 @ 10mg/ml and 0.19-IV PLA @ 100 mg/mL in pure methylene chloride prepared atroom temperature. Solution 2B: PLA-R848 @ 100 mg/ml in pure methylenechloride prepared at room temperature. Solution 2C: PLGA-R848 @ 100mg/ml in pure methylene chloride prepared at room temperature. Solution2D: PLA-PEG-R848 @ 100 mg/ml in pure methylene chloride prepared at roomtemperature. Solution 3A: PLA-PEG-nicotine @ 100 mg/ml in pure methylenechloride prepared at room temperature. Solution 4A: 0.19-IV PLA @ 100mg/mL in pure methylene chloride prepared at room temperature. Solution5A: Polyvinyl alcohol @ 50 mg/mL in de-ionized water. Solution 5B:Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.

The water in oil (W/O) primary emulsion was prepared by combiningsolution 1 and solution 2, solution 3, and solution 4 in a smallpressure tube and sonicating at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. The water/oil/water (W/O/W) doubleemulsion was prepared by adding solution 5 to the primary emulsion andsonicating at 30% to 35% amplitude for 40 seconds using the BransonDigital Sonifier 250.

The double emulsion was added to a beaker containing phosphate buffersolution (30 mL) and stirred at room temperature for 2 hours to allowfor the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and spinning at 5,000 to9,500 RPM for one hour, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeatedand the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 35 Determination Of Amount Of Agents

Method for R848 and Peptides (e.g., Ova Peptide, Human Peptide,TT2pDT5t)

The amount of R848 (immunostimulatory agent) and ova peptide (T cellantigen) was measured using reverse phase HPLC on an Agilent 1100 systemat appropriate wavelengths (λ=254 nm for R848 and 215 nm for ovapeptide) equipped with an Agilent Zorbax SB-C18 column (3.5 m. 75×4.6mm. Column Temp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA)of 95% water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA (Gradient: B=5 to 45% in 7 minutes;ramp to 95% B to 9 min; decrease back to 5% B to 9.5 min and keptequilibrating to end. Total run time was 13 minute with flow rate of 1mL/min).

Method for CpG

The amount of CpG (immunostimulatory agent) was measured using reversephase HPLC on Agilent 1100 system at 260 nm equipped with Waters XBridgeC-18 (2.5 micron particle, 50×4.6 mm ID (part No. 186003090), columntemp. 600 C) using mobile phase A of 2% acetonitrile in 100 mMTEA-acetic acid buffer, pH about 8.0 and mobile B as 90% acetonitrile,10% water (column equilibrated at 5% B, increased to 55% B in 8.5 min,then ramped to 90% B to 12 minutes. Strength of B was rapidly decreasedto 5% in one minute and equilibrated until stop time, 16 minutes. Theflow rate was 1 mL/min until end of the method, 16 minutes).

Method for Nicotine Analog

Nicotine analog was measured using reverse phase HPLC on Agilent 1100system at 254 nm equipped with Waters X-Bridge C-18 (5 micron particle,100×4.6 mm ID, column temp at 40° C.) using Mobile Phase A (MPA) of 95%water/5% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA (gradient: column was equilibrated at5% B increased to 45% B in 14 minutes. Then ramped up to 95% B from 14to 20 minutes. Mobile B strength was quickly decreased back to 5% andrequilibrated until the end of the method. The flow rate of the methodwas maintained at 0.5 ml/min with total run time of 25 minutes. The NCsuspension was centrifuged 14000 rpm for about 15-30 minutes dependingon particle size. The collected pellets were treated with 200 uL ofconc. NH₄OH (8 M) for 2 h with agitation until the solution turns clear.A 200 uL of 1% TFA was added to neutralize the mixture solution, whichbrought the total volume of the pellet solution to 200 uL. An aliquot of50 uL of the solution was diluted with MPA(or water) to 200 uL andanalyzed on HPLC as above to determine the amount present in thepellets.

Encapsulated Free R848 in Nanocarrier

0.5 mL of the NC suspension was centrifuged 14000 rpm for about 15minutes. The collected pellet was dissolved with 0.3 mL of acetonitrileand centrifuged briefly 14000 rpm to remove any residual insolubles. Theclear solution was further diluted with 4 times equivalent volume of MPAand assayed on reverse phase HPLC described above.

Encapsulated CpG in Nanocarrier

330 uL of NC suspension from the manufacture (about 10 mg/mL suspensionin PBS) was spun down at 14000 rpm for 15 to 30 minutes depending onparticle size. The collected pellets were re-suspended with 500 uL ofwater and sonicated for 30 minutes to fully disperse the particles. TheNC was then heated at 600° C. for 10 minutes. Additional 200 uL of 1 NNaOH was added to the mixture, heated for another 5 minutes where themixture becomes clear. The hydrolyzed NC solution was centrifugedbriefly at 14000 rpm. A final 2× dilution of the clear solution usingwater was then made and assayed on the reverse HPLC described above.

Encapsulated T Cell Antigens (e.g., Ova Peptide, or Human Peptide,TT2pDT5t)

330 uL of NC suspension from the manufacture (about 10 mg/mL suspensionin PBS) was spun down at 14000 rpm for 15 to 30 minutes. 100 uL ofacetonitrile was added to the pellets to dissolve the polymer componentsof the NC. The mixture was vortexed and sonicated for 1 to 5 minutes.100 uL 0.2% TFA was added to the mixture to extract the peptides andsonicated for another 5 minutes to ensure the break down of theaggregates. The mixture was centrifuged at 14000 rpm for 15 minutes toseparate any insoluble materials (e.g., polymers). A 50 uL aliquot ofthe supernatant diluted with 150 uL of MPA (or water) was taken andassayed on the reverse phase HPLC as described above.

Amount of Conjugated Nicotine Analog (B Cell Antigen) in Nanocarriers

1.5 mL of NC suspension was spun down 14000 rpm for about 15 minutes,the pellets were hydrolyzed using 150 uL of concentrated NH₄OH (8M) forabout 2-3 h until the solution turns clear. A 150 uL of 2% TFA(aq)solution was added to the pellet mixture to neutralize the solution. A100 uL aliquot of the mixture was diluted with 200 uL of water andassayed on reverse phase HPLC described above and quantified based onthe standard curve established using the precursor (PEG-nicotine) of thePLA-PEG-nicotine used in the manufacture.

Example 36 Release Rate Testing

The release of T-cell antigen, ova peptide and adjuvant, R848 from thesynthetic nanocarrier (nanoparticles) in PBS (100 mM, pH=7.4) andCitrate buffer (100 mM, pH=4.5) at 37° C. were performed as follows:

Analytical Method: The amount of R848 and ova peptide released ismeasured using reverse phase HPLC on a Agilent 1100 system at X=215 nmequipped with an Agilent Zorbax SB-C18 column (3.5 m. 75×4.6 mm. ColumnTemp=40° C. (part no. 866953-902)) using Mobile Phase A (MPA) of 98%water/2% acetonitrile/0.1% TFA and Mobile Phase B (MPB) of 90%acetonitrile/10% water/0.09% TFA with Gradient: B=5 to 45% in 7 minutes;ramp to 95% B to 9 min; re-EQ to end. 13 minute run time. Flow=1 mL/min.

The total amount of R848 and ova peptide present in the nanoparticleswas as shown in Table 1. An aqueous suspension of the tested syntheticnanocarriers was then diluted to a final stock volume of 4.4 mL withPBS.

(A) In Vitro Release Rate Measurement in PBS (pH=7.4):

For T0 sample, a 200 μL aliquot was immediately removed from each of theNP sample and centrifuged 14000 rpm in a microcentrifuge tubes using aMicrocentrifuge (Model: Galaxy 16). 100 μL of supernatant was removedand diluted to 200 μL in HPLC Mobile Phase A (MPA) and assayed for theamount of R848 and ova peptide released on the reverse phase HPLC.

For time point measurements: 9×200 μL of each of the samples were addedto microcentrifuge tubes (3×200 for unconjugated) and 300 μL of 37 C PBSwas added to each above aliquot and the samples were placed immediatelyin 37° C. oven. At the following time points: 24 hr, 48 hr, 96 hr and144 hr (for conjugated R848) or 2 h, 16 h and 24 h (for unconjugated(encapsulated) R848), the samples were centrifuged and assayed for theamount of R848 and ova peptide released as above for T0 sample.

(B) In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5):

For T0 sample, a 200 μL aliquot was removed from each of the samples andcentrifuged 6000 rpm for 20 minutes and the supernatant was removed. Theresidue nanoparticles was resuspended in 200 uL of citrate buffer andcentrifuged@14000 rpm for 15 minutes. 100 uL of the supernatant wasremoved and diluted to 200 uL with MPA and assayed for R848 and peptideas above.

For time point measurements: 9×200 uL of each of the samples were addedto microcentrifuge tubes (3×200 for unconjugated) and centrifuged for 20minutes@6000 rpm and the supernatants were removed. The residue NPs werethen resuspended in 500 uL of citrate buffer and placed in 37° C. oven.At the following time points: 24 hr, 48 hr, 96 hr and 144 hr (forconjugated R848) or 2 h, 16 h and 24 h (for unconjugated (encapsulated)R848), the samples were centrifuged and assayed for the amount of R848and ova peptide released as above for T0 sample.

In order to complete the mass balance from above measurements in PBS andCitrate buffer, the remaining pellets (conjugated R848 samples only)from each sample was treated with 200 uL of conc. NH4OH (8 M) for 3 hwith mixing. After the mixture was settled, 200 uL of 1% TFA was addedto bring total volume of the pellet to 400 uL. An aliquot of 50 uL ofthe solution was diluted with MPA to 200 uL and analyzed on HPLC asabove to determine the amount of R848 and ova peptide that remained inthe pellet after in vitro release to close the mass balance. Forunconjugated samples, the sample was diluted with TFA in acetonitrileand assayed as above for R848 and peptide.

The results are summarized in FIGS. 1-3.

Materials and Method—

HPLC—Agilent 1100. λ=215 nm. Column Temp=40° C.

Column—Agilent Zorbax SB-C18, 3.5 μm. 75×4.6 mm. (part no. 866953-902)

C18 guard column

Mobile Phase A (MPA)-98% water/2% acetonitrile/0.1% TFA

Mobile Phase B (MPB)-90% acetonitrile/10% water/0.09% TFA

-   -   Gradient: B=5 to 45% in 7 minutes; ramp to 95% B to 9 min; re-EQ        to end. 13 minute run time. Flow=1 mL/min.

PBS—100 mM, pH=7.4.

Citrate Buffer—100 mM, pH=4.5.

Oven—

Microcentrifuge—Galaxy 16

Microcentrifuge tubes

Sonicator

Pipets—20, 200, 1000 μL adjustable

HPLC grade water—EMD-#WX0008-1.

NH₄OH—˜8M. Mallinkcrodt.

TFA, 0.2%. Prep Apr. 27, 2009.

TFA, 1%. Prep May 13, 2009.

Thermometer

Samples

“6-1” and “6-2” have entrapped R848. All of the rest have conjugatedR848. The estimated values are based on the loading results from the“62” series.

TABLE 2 Estimated R848 and Ova peptide in synthetic nanocarriers:Estimated R848 in Estimated Ova in Sample ID NPs (μg/mL) NPs (μg/mL) 154 146 2 166 184 3 119 32 4 114 34 5 465 37 6 315 34 7 116 40

Sample volumes were slightly below what was planned. To ensure enoughmaterial is available for all time points, the following volumes of PBSwere added to the samples to bring them all to 4.4 mL.

TABLE 3 Sample Volume PBS added Sample ID Volume (mL) (mL) 1 4.35 0.05 24.23 0.17 3 4.21 0.19 4 4.20 0.20 5 4.21 0.19 6 4.19 0.21 7 4.20 0.20

Procedure—

-   -   1) T=0 Sample Prep        -   a. PBS            -   i. Remove a 200 μL aliquot from each of the samples.                Microcentrifuge@14000 rpm. Remove supernatant.            -   ii. Dilute supernatant 100 μL≧200 μL in MPA. (DF=2).            -   iii. Assay for peptide and R848.        -   b. Citrate            -   i. Remove a 200 μL aliquot from each of the samples.                Microcentrifuge@6000 rpm for 20 minutes. Remove                supernatant.            -   ii. Add 200 uL of citrate buffer and thoroughly                resuspend.            -   iii. Microcentrifuge@14000 rpm for 15 minutes. Remove                supernatant.            -   iv. Dilute supernatant 100 μL≧200 μL in MPA. (DF=2)            -   v. Assay for peptide and R848.    -   2) PBS IVR        -   a. Add 9×200 μL of each of the samples to microcentrifuge            tubes. (3×200 for unconjugated)        -   b. To each aliquot add 300 μL of 37 C PBS.        -   c. Immediately place samples in 37 C oven.    -   3) Citrate IVR        -   a. Add 9×200 uL of each of the samples to microcentrifuge            tubes. (3×200 for unconjugated)        -   b. Centrifuge for 20 minutes 6000 rpm.        -   c. Remove the supernatants.        -   d. To each tube, add 500 μL of citrate buffer and resuspend            thoroughly.        -   e. Place samples in 37 C oven    -   4) For lots 1-4 and 8, remove the samples (see step 6) at the        following time points:        -   a. Conjugated            -   i. 24 hr            -   ii. 48 hr (2 days)            -   iii. 96 hr (4 days)            -   iv. 144 hr (6 days)            -   v. Further time points TBD based on the above data.        -   b. Non conjugated            -   i. 2 hr            -   ii. 16 hr            -   iii. 24 hr    -   5) For lots 6 and 7, remove samples at the following time        points:        -   a. PBS            -   i. 24 hr            -   ii. 48 hr (2 days)            -   iii. 96 hr (4 days)            -   iv. 144 hr (6 days)            -   v. Further time points TBD based on the above data.        -   b. Citrate            -   i. 2 hr            -   ii. 16 hr            -   iii. 24 hr            -   iv. 48 hr (2 days)            -   v. 72 hr (3 days)            -   vi. 96 hr (4 days)            -   vii. 120 hr (5 days)            -   viii. Further time points TBD based on the above data.        -   6) Sample as follows:        -   a. Microcentrifuge@14000 rpm for 15 minutes.        -   b. Remove supernatant.        -   c. Dilute 100 μL to 200 μL in MPA. (DF=2)    -   7) Assay for peptide and R848. This will provide the amount        released at each time point.

To Complete Mass Balance, Perform the Following:

-   -   8) To the remaining pellets (conjugated only) add 200 uL NH₄OH.    -   9) Vortex briefly and sonicate to disperse.    -   10) Add stir bar. Allow to sit until clear (at least 3 hours).    -   11) Add 200 uL of 1% TFA (total pellet volume=400 μL).    -   12) Dilute 50 μL to 200 μL in MPA. Analyze by HPLC to determine        peptide and R848 remaining in the pellet. (DF=4).    -   13) For unconjugated lots, assay for peptide and R848 with        typical AcN/TFA method.

Example 37 Release Rate Testing

The release of antigen (e.g., ova peptide, T cell antigen) andimmunostimulatory agents (e.g., R848, CpG) from synthetic nanocarriersin phosphate buffered saline solution (PBS) (100 mM, pH=7.4) and citratebuffer (100 mM, pH=4.5) at 37° C. was determined as follows:

The release of R848 from the nanocarrier composed of conjugated R848 andthe ova peptide was achieved by exchanging desired amount of the aqueoussuspension of the tested synthetic nanocarriers obtained from themanufacture (e.g., about 10 mg/mL in PBS) into the same volume of theappropriate release media (Citrate buffer 100 mM) via centrifugation andre-suspension.

In Vitro Release Rate Measurement in PBS (pH=7.4)

1 mL of the PBS suspension NC was centrifuged 14000 rpm inmicrocentrifuge tubes generally from 15-30 minutes depending on particlesize. The collected supernatant was then diluted with equal volume ofthe mobile phase A (MPA) or water and assayed on reverse phase HPLC forthe amount of the R848 release during the storage. The remaining pelletwas re-suspended to homogeneous suspension in 1 mL of PBS and placed to37° C. thermal chamber with constant gentle agitation

For T0 sample, a 150 μL aliquot was immediately removed from NCsuspension prior placing the NC suspension to 37° C. thermal chamber andcentrifuged 14000 rpm in microcentrifuge tubes using a microcentrifuge(Model: Galaxy 16). 100 L of the supernatant was removed and diluted to200 μL with HPLC Mobile Phase A (MPA) or water and assayed for theamount of R848 and ova peptide released on the reverse phase HPLC.

For time point measurements, 150 μL aliquot was removed from the 37° C.NC sample suspension, and the samples were centrifuged and assayed forthe amount of R848 and ova peptide released in the same manner as for T0sample. The R848 and ova peptide released was tested at 6 h, 24 h forroutine monitoring with additional 2 h, 48 h, 96 h and 144 h forcomplete release profile establishment.

In Vitro Release Rate Measurement in Citrate Buffer (pH=4.5)

A 100 mM sodium citrate buffer (pH=4.5) was applied to exchange theoriginal NC storage solution (e.g., PBS) instead of the PBS buffer,pH=7.4. In order to complete the mass balance from above measurements inPBS and Citrate buffer, the remaining pellets from each time point weretreated with 100 uL of NH₄OH (8 M) for 2 h (or more) with agitationuntil solution turn clear. A 100 uL of 1% TFA was added to neutralizethe mixture, which brought the total volume of the pellet solution to200 uL. An aliquot of 50 uL of the mixture was diluted with MPA (orwater) to 200 uL and analyzed on HPLC as above to determine the amountof unreleased R848 remaining in the pellets after in vitro release toclose the mass balance. For unconjugated samples, the sample was dilutedwith TFA in acetonitrile and assayed as above for R848.

The release of CpG was determined similar to the measurement of R848 andova peptide in terms of sample preparation and monitored time points.However, the amount of the CpG in the release media was assayed by thereverse phase HPLC method described above.

Example 38 Immunization with NC-Nic Carrying CpG Adjuvant

Groups of five mice were immunized three times (subcutaneously, hindlimbs) at 2-week intervals (days 0, 14 and 28) with 100 μg of NC-Nic.NC-Nic was a composition of nanocarriers exhibiting nicotine on theouter surface and, for all groups of mice except for Group 1, carryingCpG-1826 (thioated) adjuvant, which was released from the nanocarriersat different rates. The nanocarriers were prepared according to a methodprovided above. Serum anti-nicotine antibodies were then measured ondays 26 and 40. EC₅₀ for anti-nicotine antibodies as measured instandard ELISA against polylysine-nicotine are shown in FIG. 4.

The Group 1 mice were administered NC-Nic w/o CpG-1826 containing Ovapeptide and polymers, 75% of which were PLA and 25% were PLA-PEG-Nic.The Group 2 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 3.2%CpG-1826; release rate at 24 hours: 4.2 g CpG per mg of NC. The Group 3mice were administered NC-Nic containing polymers, 75% of which were PLAand 25% were PLA-PEG-Nic, and 3.1% CpG-1826; release rate at 24 hours:15 g CpG per mg of NC. Release was determined at a pH of 4.5.

The results shown in FIG. 4 demonstrate that entrapment of adjuvant intonanocarriers is beneficial for the immune response against NC-associatedantigen, and, furthermore, that the higher release rate of entrapped CpGadjuvant from within the nanocarriers (NC) at 24 hours produced animmune response, which was elevated compared to one induced by NC with aslower release rate of CpG adjuvant (a TLR9 agonist).

Example 39 Immunization with NC-Nic Carrying Two Forms of CpG Adjuvant

Groups of five mice were immunized two times (subcutaneously, hindlimbs) at 4-week intervals (days 0, and 28) with 100 μg of NC-Nic andserum anti-nicotine antibodies were then measured on days 12, 24 and 40.NC-Nic was a composition of nanocarriers exhibiting nicotine on theouter surface and carrying one of two forms of CpG-1826 adjuvant. Thenanocarriers were prepared according to a method provided above. EC₅₀for anti-nicotine antibodies as measured in standard ELISA againstpolylysine-nicotine are shown in FIG. 5.

The Group 1 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 6.2%CpG-1826 (thioated); release rate at 24 hours: 16.6 μg CpG per mg of NC.The Group 2 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.2%CpG-1826 (thioated); release rate at 24 hours: 13.2 μg CpG per mg of NC.The Group 3 mice were administered NC-Nic containing ova peptide,polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and 7.9%CpG-1826 (phosphodiester or PO, non-thioated); release rate at 24 hours:19.6 μg CpG per mg of NC. The Group 4 mice were administered NC-Niccontaining ova peptide, polymers, 75% of which were PLA and 25% werePLA-PEG-Nic, and 8.5% CpG-1826 (PO, non-thioated); release rate at 24hours: 9.3 μg CpG per mg of NC. Release was determined at a pH of 4.5.

The results shown in FIG. 5 demonstrate that the rate of release ofentrapped adjuvant (CpG, TLR9 agonist) from nanocarriers influencedproduction of an antibody to NC-bound antigen (nicotine) with thenanocarrier exhibiting higher release rate at 24 hours induced strongerhumoral immune response (group 1>group 2 and group 3>group 4). This wastrue irrespective of CpG form used (more stable, thioated or less stablenon-thioated).

Example 40 Immunization with NC-Nic Carrying R848

Groups of five mice were immunized three times (subcutaneously, hindlimbs) at 2-week intervals (days 0, 14 and 28) with 100 μg of NC-Nic andserum anti-nicotine antibodies were then measured on days 26, 40 and 54.The nanocarriers were prepared according to a method provided above.EC₅₀ for anti-nicotine antibodies as measured in standard ELISA againstpolylysine-nicotine are shown in FIG. 6.

The Group 1 mice were administered NC-Nic containing ova peptide andpolymers, 75% of which were PLA and 25% were PLA-PEG-Nic, but withoutadjuvant. The Group 2 mice were administered NC-Nic containing ovapeptide, polymers, 75% of which were PLA and 25% were PLA-PEG-Nic, and1.0% R848; of which 92% is released at 2 hours and more than 96% isreleased at 6 hours. The Group 3 mice were administered NC-Niccontaining ova peptide, polymers, 75% of which were PLA-R848 and 25%were PLA-PEG-Nic, and 1.3% R848, of which 29.4% is released at 6 hoursand 67.8% is released at 24 hours. The Group 4 mice were administeredNC-Nic containing ova peptide, polymers, 75% of which were PLA-R848 and25% were PLA-PEG-Nic, and 1.4% of R848, of which 20.4% is released at 6hours and 41.5% is released at 24 hours. The Group 5 mice wereadministered NC-Nic containing ova peptide, polymers, 25% of which werePLA-PEG-R848, 50% PLA, and 25% were PLA-PEG-Nic, and 0.7% of R848; ofwhich less than 1% is released at 24 hours. Release was determined at apH of 4.5.

The results shown in FIG. 6 demonstrate that R848 adjuvant (a TLR 7/8agonist) contained in the NC augments humoral immune response againstNC-associated antigen (groups 2-5≧≧group 1). Furthermore, neither fast(group 2), nor slow (group 5) release of R848 was elevated an immuneresponse to the same level as NC releasing R848 at intermediate rate(group 3≈group 4≈group 2≈group 5).

Example 41 Immunization with NC-Nic Carrying Entrapped PO CpG

Groups of five mice were immunized three times (subcutaneously, hindlimbs) at 2-week intervals (days 0, 14 and 28) with 100 μg of NC-Nic(nanocarrier exhibiting nicotine on the outer surface) with entrappedPO-CpG or not containing entrapped PO-CpG admixed with free PO-CpG. Thesynthetic nanocarriers were prepared according to methods providedabove. Serum anti-nicotine antibodies were then measured in both groupson days 26 and 40. EC₅₀ for anti-nicotine antibodies as determined instandard ELISA against polylysine-nicotine are shown in FIG. 7.

The group 1 mice were immunized with a NC-Nic with 1826 PO-CpG andMHC-II helper peptide from ovalbumin (Ov-II) encapsulated (6.6% PO-CpG;2.3% Ov-II). The group 2 mice were immunized with a NC-Nic with 0.7% ofentrapped Ov-II admixed with 20 μg of free 1826 PO-CpG.

This experiment demonstrates that the entrapment of PO-CpG within thenanocarrier (NC) generates a humoral immune response, which was superiorto one induced when a ˜3-fold higher dose of free PO-CpG is admixed toNC without entrapped PO-CpG (antibody titer in group 1>antibody titer ingroup 2).

1. A method, comprising: determining that immunomodulatory agentscoupled to synthetic nanocarriers dissociate from the syntheticnanocarriers according to the following relationship:IArel(4.5)24%/IArel(7.4)24%≧1.2, wherein IArel(4.5)24% is defined as aweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hoursdivided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=4.5 for 24 hours plus a weight of immunomodulatory agentretained in the synthetic nanocarrier upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=4.5 for 24 hours,expressed as weight percent, and taken as an average across a sample ofthe synthetic nanocarriers, and wherein IArel(7.4)24% is defined as aweight of immunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hoursdivided by the sum of the weight of immunomodulatory agent released uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=7.4 for 24 hours plus a weight of immunomodulatory agentretained in the synthetic nanocarrier upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hours,expressed as weight percent, and taken as an average across a sample ofthe synthetic nanocarriers; and causing the synthetic nanocarriers to beadministered to a subject.
 2. A method, comprising: causing the releaseof immunomodulatory agents in a subject where the immunomodulatoryagents are coupled to synthetic nanocarriers and determined todissociate from the synthetic nanocarriers according to the followingrelationship: IArel(4.5)24%/IArel(7.4)24%≧1.2, wherein IArel(4.5)24% isdefined as a weight of immunomodulatory agent released upon exposure ofthe synthetic nanocarrier to an in vitro aqueous environment at a pH=4.5for 24 hours divided by the sum of the weight of immunomodulatory agentreleased upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=4.5 for 24 hours plus a weight ofimmunomodulatory agent retained in the synthetic nanocarrier uponexposure of the synthetic nanocarrier to an in vitro aqueous environmentat a pH=4.5 for 24 hours, expressed as weight percent, and taken as anaverage across a sample of the synthetic nanocarriers, and whereinIArel(7.4)24% is defined as a weight of immunomodulatory agent releasedupon exposure of the synthetic nanocarrier to an in vitro aqueousenvironment at a pH=7.4 for 24 hours divided by the sum of the weight ofimmunomodulatory agent released upon exposure of the syntheticnanocarrier to an in vitro aqueous environment at a pH=7.4 for 24 hoursplus a weight of immunomodulatory agent retained in the syntheticnanocarrier upon exposure of the synthetic nanocarrier to an in vitroaqueous environment at a pH=7.4 for 24 hours, expressed as weightpercent, and taken as an average across a sample of the syntheticnanocarriers.
 3. The method of claim 1, wherein the immunomodulatoryagents are coupled to the synthetic nanocarriers via immunomodulatoryagent coupling moieties.
 4. The method of claim 1, wherein theimmunomodulatory agents are encapsulated within the syntheticnanocarriers.
 5. The method of claim 1, wherein the immunomodulatoryagents comprise an adjuvant.
 6. The method of claim 5, wherein theadjuvant comprises a Toll-like receptor (TLR) agonist.
 7. The method ofclaim 6, wherein the TLR agonist is a TLR 3 agonist, TLR 7 agonist, TLR8 agonist, TLR 7/8 agonist, or a TLR 9 agonist.
 8. The method of claim6, wherein the TLR agonist comprises an immunostimulatory nucleic acid,an imidazoquinoline, or an adenine derivative.
 9. The method of claim 8,wherein the imidazoquinoline comprises an imidazoquinoline amine, animidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, animidazoquinoline amine, imiquimod, or resiquimod.
 10. The method ofclaim 1, wherein the synthetic nanocarriers further comprise a B cellantigen and/or a T cell antigen.
 11. The method of claim 1, wherein thesynthetic nanocarriers comprise lipid-based nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles, peptide orprotein-based particles, nanoparticles that comprise a combination ofnanomaterials, spheroidal nanoparticles, cubic nanoparticles, pyramidalnanoparticles, oblong nanoparticles, cylindrical nanoparticles, ortoroidal nanoparticles.
 12. The method of claim 11, wherein thesynthetic nanocarriers comprise polymeric nanoparticles.
 13. The methodof claim 12, wherein the polymeric nanoparticles comprise one or morebiodegradable polymers.
 14. The method of claim 13, wherein thebiodegradable polymers comprise poly(lactide), poly(glycolide), orpoly(lactide-co-glycolide).
 15. The method of claim 13, wherein theimmunomodulatory agents are coupled to the one or more biodegradablepolymers via immunomodulatory agent coupling moieties.
 16. The method ofclaim 15, wherein the immunomodulatory agent coupling moieties comprisean amide bond.
 17. The method of claim 15, wherein the immunomodulatoryagent coupling moieties comprise an ester bond.
 18. The method of claim1, wherein the synthetic nanocarriers are administered to the subject.19. The method of claim 1, wherein an immune response is induced orenhanced in the subject.
 20. The method of claim 1, wherein the subjecthas cancer, an infectious disease, a non-autoimmune metabolic disease, adegenerative disease, or an addiction.